AU2018203206A1 - Factor VIII Complex With XTEN and Von Willebrand Factor Protein, and Uses Thereof - Google Patents

Abstract

The present invention includes a chimeric protein comprising a VWF protein with D' domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain of a VWF fragment is associated with a FVIII polypeptide chain linked to an XTEN sequence. The VWF fragment polypeptide chain can prevent or inhibit binding of endogenous VWF to FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to FVIII protein, VWF fragment can extend half-life of chimeric protein comprising FVIII protein. The invention includes nucleotides, vectors, host cells, use of VWF fragment, or chimeric proteins.

Description

The present invention includes a chimeric protein comprising a VWF protein with D' domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain of a VWF fragment is associated with a FVIII polypeptide chain linked to an XTEN sequence. The VWF fragment polypeptide chain can prevent or inhibit binding of endogenous VWF to FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to FVIII protein, VWF fragment can extend half-life of chimeric protein comprising FVIII protein. The invention includes nucleotides, vectors, host cells, use of VWF fragment, or chimeric proteins.

2018203206 08 May 2018

FACTOR VIII COMPLEX WITH XTEN AND VON WILLEBRAND FACTOR PROTEIN,

AND USES THEREOF [0001] This application is a divisional of Australian Patent Application No.

2013290173, filed on 10 July 2013, and is related to International Patent Application No. PCT/US2013/049989, filed on 10 July 2013, and claims priority from U.S. Provisional Patent Application Nos. 61/670,401 filed on 11 July 2012, 61/759,819 filed on 1 February 2013, 61/801,504 filed on 15 March 2013, 61/801,544 filed on 15 March 2013, 61/827,158 filed on 24 May 2013, and 61/840,811 filed on 28 June 2013, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION [0001a] Haemophilia A is a bleeding disorder caused by defects in the gene encoding coagulation factor VIII (FVIII) and affects 1-2 in 10,000 male births. Graw et al., Nat. Rev. Genet. 6(6): 488-501 (2005). Patients affected with hemophilia A can be treated with infusion of purified or recombinantly produced FVIII. All commercially available FVIII products, however, are known to have a half-life of about 8-12 hours, requiring frequent intravenous administration to the patients. See Weiner M.A. and Cairo, M.S., Pediatric Hematology Secrets, Lee, M.T., 12. Disorders of Coagulation, Elsevier Health Sciences, 2001; Lillicrap, D. Thromb. Res. 122 Suppl 4:S2-8 (2008). In addition, a number of approaches have been tried in order to extend the FVIII half-life. For example, the approaches in development to extend the half-life of clotting factors include pegylation, glycopegylation, and conjugation with albumin. See Dumont et al., Blood. 119(13): 3024-3030 (Published online Jan. 13, 2012). Regardless of the protein engineering used, however, the long acting FVIII products currently under development are reported to have limited half-lives - only to about 1.5 to 2 hours in preclinical animal models. See id. Consistent results have been demonstrated in humans, for example, rFVIIIFc was reported to improve half-life up to ~ 1.7 fold compared with ADVATE® in hemophilia A patients. See Id. Therefore, the half-life increases, despite minor improvements, may indicate the presence of other Tl/2 limiting factors. See Liu, T. et al., 2007 ISTH meeting, abstract #PM- 035; Henrik, A. et al., 2011 ISTH meeting, abstract #P=MO-181; Liu, T. et al., 2011 ISTH meeting abstract #P-WE-131.

[0002] Plasma von Willebrand Factor (VWF) has a half-life of approximately 12 hours (ranging from 9 to 15 hours).

-1 http://www.nhlbi.nih.gov/guidelines/vwd/2_scientificoverview.htm (last visited October 22,

2011). The VWF half-life may be affected by a number of factors: glycosylation pattern,

ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin motif- 13), and various mutations in VWF.

[0003] In plasma, 95-98% of FVIII circulates in a tight non-covalent complex with full length VWF. The formation of this complex is important for the [Text continues on page 2,]

2018203206 08 May 2018

- la 2018203206 08 May 2018 maintenance of appropriate plasma levels of FVIII in vivo. Lenting et al., Blood. 92(11): 3983-96 (1998); Lenting et al., J. Thromb. Haemost. 5(7): 1353-60 (2007). The full-length wild-type FVIII is mostly present as a heterodimer having a heavy chain (MW 200kD) and a light chain (MW 73kD). When FVIII is activated due to proteolysis at positions 372 and 740 in the heavy chain and at position 1689 in the light chain, the VWF bound to FVIII is removed from the activated FVIII. The activated FVIII, together with activated factor IX, calcium, and phospholipid (tenase complex), induces the activation of factor X, generating large amounts of thrombin. Thrombin, in turn, then cleaves fibrinogen to form soluble fibrin monomers, which then spontaneously polymerize to form the soluble fibrin polymer. Thrombin also activates factor XIII, which, together with calcium, serves to crosslink and stabilize the soluble fibrin polymer, forming crosslinked (insoluble) fibrin. The activated FVIII is cleared fast from the circulation by proteolysis.

[0004] Due to the frequent dosing and inconvenience caused by the dosing schedule, there is still a need to develop FVIII products requiring less frequent administration, i.e., a FVIII product that has a half-life longer than the 1.5 to 2 fold half-life limitation.

BRIEF SUMMARY OF THE INVENTION [0005] The present invention is directed to a chimeric protein comprising (i) a von

Willebrand Factor (VWF) fragment comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker and wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein. The chimeric protein can comprise a single polypeptide chain comprising the VWF fragment, the XTEN sequence, and the FVIII protein, or two polypeptide chains, a first chain comprising the VWF fragment and the second chain comprising the FVIII protein, wherein the XTEN polypeptide is linked either to the VWF fragment or the FVIII protein.

[0006] In one embodiment, the chimeric protein of the invention comprises a formula comprising:

(a) V-X-FVIII,

-22018203206 08 May 2018 (b) FVIII-X-V, (c) V-X:FVIII, (d) X-V:FVIII, (e) FVIII:V-X, or (f) FVIII:X-V, wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences, and

FVIII comprises a FVIII protein. The hyphen (-) can be a peptide bond or a linker, e.g., a cleavable linker, while the colon (:) represents a chemical association or a physical association between the polypeptides, for example a covalent or non-covalent bond.

[0007] In another embodiment, the chimeric protein further comprises (iv) an immunoglobulin (Ig) constant region or a portion thereof (also indicated as FI or a first Ig constant region or a portion thereof) linked to the VWF fragment, the XTEN sequence, the FVIII protein, or any combinations thereof. In other embodiments, the chimeric protein further comprises an additional Ig constant region or a portion thereof (also indicated as F2 or a second Ig constant region or a portion thereof). The first Ig constant region or a portion thereof can be linked to the VWF fragment or the XTEN sequence, and the second Ig constant region can be linked to the FVIII protein. The first Ig constant region, the second Ig constant region or a portion thereof, or both can extend the half-life of the FVIII protein.

[0008] In some embodiments, the second Ig constant region or a portion thereof (F2) is linked to the VWF fragment by a linker, e.g., a processable linker. In other embodiments, the second Ig constant region or a portion thereof (F2) is associated with the (first) Ig constant region or a portion thereof (FI). The second Ig constant region or a portion thereof (F2) and the first Ig constant region or a portion thereof (FI) can be identical or different. The second Ig constant region or a portion thereof can be associated with the Ig constant region or a portion thereof by a covalent bond, e.g., a disulfide bond. The VWF fragment linked to the first Ig constant region or a portion thereof may also be associated with the FVIII protein linked to the second Fc region by a non-covalent bond. In certain embodiments, the FVIII protein can further comprise one or more additional XTEN sequences which are linked to the C-terminus or N-terminus of the FVIII

-3 2018203206 08 May 2018 protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites). In some embodiments, the half-life of the FVIII protein is extended, compared to wild type FVIII or a FVIII protein without the VWF fragment.

[0009] In some embodiments, the chimeric protein comprises a formula comprising:

(g) V-L2-X-L1-F1:FVIII-L3-F2;

(h) V-L2-X-L1-F1:F2-L3-FVIII;

(i) F1-L1-X-L2-V:FVIII-L3-F2;

(j) F1-L1-X-L2-V:F2-L3-FVIII;

(k) V-L2-X-L1-F1-L4-FVIII-L3-F2;

(l) F2-L3-FVIII-L4-F1-L1-X-L2-V;

(m) FVIII-L3-F2-L4-V-L2-X-L1-F1; or (n) F1-L1-X-L2-V-L4-F2-L3-FVIII, wherein V comprises a VWF fragment, each of Ll, L2, and L3 comprises an optional linker, e.g., a cleavable linker,

L4 is an optional linker, e.g., a processable linker,

FVIII comprises a FVIII protein,

X comprises one or more XTEN sequences,

FI comprises an optional first Ig constant region or a portion thereof,

F2 comprises an optional second Ig constant region or a portion thereof, and (:) is a covalent bond or non-covalent bond.

[0010] The present invention is also directed to a chimeric protein comprising (i) a

FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more insertion sites) and wherein the Ig constant region or a portion thereof is linked to or associated with the FVIII protein or the XTEN sequence. In one embodiment, the Ig constant region or a portion thereof useful for the chimeric protein comprises a first Fc region. In another embodiment, the chimeric protein further comprises an additional Ig constant region or a portion thereof. The additional Ig constant region or a portion thereof useful for the invention can

-42018203206 08 May 2018 comprise a second Fc region, which is linked to or associated with the first Fc region, e.g., by a covalent bond. In other embodiments, the first Fc region is linked to the second Fc region by a linker, e.g., a processable linker.

[0011] In other aspects, a chimeric protein comprises (i) a FVIII protein, (ii) an

XTEN sequence, (iii) a VWF fragment, and (iv) an lg constant region or a portion thereof, which comprises the D' domain and the D3 domain of VWF, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the Nterminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more insertion sites), the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the lg constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof. Non-limiting examples of the chimeric proteins may comprise a formula, which comprises:

(1) F VIII(X 1 )-L 1 -F1: V-L2-X2-L3 -F2;

(2) FVIII(X1)-L1-F1 :F2-L3-X2-L2-V;

(3) F1 -L1 -FVIII(X 1): V-L2-X2-L3 -F2;

(4) F1 -L1 -FVIII(X 1) :F2-L3 -X2-L2-V;

(5) FVIII(X1 )-L 1 -F1-L4-V-L2-X2-L3-F2;

(6) FVIII(X1 )-Ll -F1-L4-F2-L3-X2-L2-V;

(7) F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (8) F1 -L1 -FVIII(X1 )-L4-F2-L3-X2-L2-V, wherein FVIII(Xl) comprises a FVIII protein and one or more XTEN sequences, wherein one or more of the XTEN sequences are linked to the N-terminus or Cterminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites); each of LI, L2, or L3 comprises an optional linker, e.g., a cleavable linker;

L4 is a linker, a processable linker;

X2 comprises one or more XTEN sequences;

FI comprises an lg constant region or a portion thereof;

F2 comprises an optional additional lg constant region or a portion thereof, and V comprises a VWF fragment;

(-) is a peptide bond or one or more amino acids; and

-5 2018203206 08 May 2018 (:) comprises a covalent bond or a non-covalent bond.

[0012] One aspect of the invention is that the VWF fragment useful for the chimeric protein does not bind to a VWF clearance receptor, which prevents or inhibits interaction of the FVIII protein with endogenous VWF. The chimeric protein comprising the VWF fragment thus has reduced clearance or is not cleared through a VWF clearance pathway. Another aspect of the invention is that the VWF fragment is capable of protecting the FVIII protein from one or more protease cleavages, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors.

[0013] Because of the VWF fragment's ability to prevent or inhibit interaction between the FVIII protein and endogenous VWF, the half-life of the FVIII protein, is extended compared to a FVIII protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0014] The Ig constant region or a portion thereof useful for the chimeric protein comprises a first Fc region, which is linked to the VWF fragment by an optional linker, e.g., a cleavable linker. The chimeric protein can further comprise an additional Ig constant region or a portion thereof, which is linked to the FVIII protein or the XTEN sequence, the Ig constant region or a portion thereof, the VWF fragment, or any combinations thereof by an optional linker. In one embodiment, the additional Ig constant region or a portion thereof is linked to the

-62018203206 08 May 2018

FVIII protein by an optional linker. The additional Ig constant region or a portion thereof can comprise a second Fc region.

[0015] The Ig constant region or a portion thereof useful in the present invention and the additional Ig constant region or a portion thereof useful in the present invention are identical or different.

[0016] In some aspects, the FVIII protein is linked to an XTEN sequence at the Cterminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in mature native human FVIII (e.g., one or more insertion sites) or any combinations thereof. One or more insertion sites in the FVIII protein can be located within one or more domains of the FVIII protein selected from the group consisting of the Al domain, the al acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the Cl domain, the C2 domain, and any combinations thereof or between one or more domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and Cl domain, the Cl domain and C2 domain, and any combinations thereof or between two domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and C1 domain, the C1 domain and C2 domain, and any combinations thereof.

[0017] In one embodiment, the one or more insertion sites are located immediately downstream of one or more amino acids in mature native human FVIII (e.g., SEQ ID NO: 4 [mature FVIII sequence-full length]) selected from the group consisting of the amino acid residues in Table 7, 8, 9, 10, 11, or any combinations thereof.

[0018] In another embodiment, the one or more insertion sites are located in one or more permissive loops of mature native human FVIII. In other embodiments, the one or more insertion sites are located in the a3 region of mature native human FVIII. For example, an XTEN sequence can be inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 (full length mature FVIII). In other embodiments, a FVIII protein is linked to at least two XTEN sequences, a

-72018203206 08 May 2018 first XTEN sequence inserted within the a3 region, and a second XTEN sequence inserted within a permissive loop in the FVIII protein (e.g., Al-1, Al-2, A2-1, A2 2, A3-1, or A3-2). In still other embodiments, a FVIII protein is linked to at least three XTEN sequences, a first XTEN sequence inserted within the a3 region and a second XTEN sequence and a third XTEN sequence inserted within one or two permissive loop in the FVIII protein (e.g., Al-1, Al-2, A2-1, A2-2, A3-1, or A32)· [0019] In certain embodiments, the one or more insertion sites for one or more

XTEN insertions are immediately downstream of one or more amino acids (numbered relative to mature FVIII sequence) selected from the group consisting

of: (1) amino acid 3,	(2) amino acid 18,	(3) amino acid 22,
(4) amino acid 26,	(5) amino acid 32,	(6) amino acid 40,
(7) amino acid 60,	(8) amino acid 65,	(9) amino acid 81,
(10) amino acid 116,	(11) amino acid 119,	(12) amino acid 130,
(13) amino acid 188,	(14) amino acid 211,	(15) amino acid 216,
(16) amino acid 220,	(17) amino acid 224,	(18) amino acid 230,
(19) amino acid 333,	(20) amino acid 336,	(21) amino acid 339,
(22) amino acid 375,	(23) amino acid 399,	(24) amino acid 403,
(25) amino acid 409,	(26) amino acid 416,	(26) amino acid 442,
(28) amino acid 487,	(29) amino acid 490,	(30) amino acid 494,
(31) amino acid 500,	(32) amino acid 518,	(33) amino acid 599,
(34) amino acid 603,	(35) amino acid 713,	(36) amino acid 745,
(37) amino acid 1656,	(38) amino acid 1711,	(39)
amino acid 1720, (40) amino acid 1725,	(41) amino acid 1749,	(42)
amino acid 1796, (43) amino acid 1802,	(44) amino acid 1827,	(45)
amino acid 1861, (46) amino acid 1896,	(47) amino acid 1900,	(48)
amino acid 1904, (49) amino acid 1905,	(50) amino acid 1910,	(51)

amino acid 1937,

-82018203206 08 May 2018 (53) amino acid 2068, (54) (52) amino acid 2019, amino acid 2111, (55) amino acid 2120, (56) amino acid 2171, (57) amino acid 2188, (58) amino acid 2227, (59) amino acid 2277, and (60) two or more combinations thereof.

[0020] In some embodiments, one XTEN is inserted in the FVIII protein. In some embodiments, two XTENs are inserted in the FVIII protein. In some embodiments, 3 XTENs are inserted in the FVIII protein.

[0021] In a particular example, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4 (full-length mature FVIII). In another example, a first XTEN is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In some examples, a first XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In other examples, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In yet other embodiments, a first XTEN is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In still other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4. In certain embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4 (full-92018203206 08 May 2018 length mature FVIII), a second XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 2, a third XTEN is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a fourth XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In another example, an XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4. In an additional example, a first XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a second XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In another example, a first XTEN is immediately inserted downstream of amino acid 403 corresponding to SEQ ID NO: 4 and a second XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 745 of corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 4, and a second XTEN is inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4.

[0022] In some embodiments, the FVIII protein is a dual chain FVIII isoform. In some embodiments, the FVIII protein is a single chain FVIII isoform.

[0023] In some embodiments, the XTEN that is inserted is SEQ ID NO: 39 (AE288). In some examples, the XTENs that are inserted are SEQ ID NOs: 38 and 37 (AG144 and AE144). In some examples, the XTENs that are inserted are

SEQ ID NOs: 37, 38 and 37 (AE144, AG144, and AE144). In some

- 102018203206 08 May 2018 embodiments, the XTENs that are inserted are SEQ ID NOs: 37 and 40 (AE144 and AE288). In some embodiments, the XTENs that are inserted are AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144 2A (SEQ IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO:

131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 39), AE288 2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG144 (SEQ ID NO: 38), and any combinations thereof.

[0024] The FVIII protein useful in the invention can comprise B domain or a portion thereof, e.g., SQ B domain deleted FVIII. In one embodiment, the FVIII protein comprises single chain FVIII. In another embodiment, the single chain FVIII contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). In other embodiments, the amino acid substitution is an amino acid other than arginine. In some embodiments, the FVIII protein comprises a heavy chain of FVIII and a light chain of FVIII, wherein the heavy chain and the light chain are associated with each other by a metal bond.

[0025] The FVIII protein can have a low affinity to or does not bind to a lowdensity lipoprotein receptor-related protein (LRP), e.g., by containing at least one amino acid substitution that lowers the affinity to or eliminates the binding to the LRP. Such at least one amino acid substitution can be at a residue corresponding to residue 471, residue 484, residue 487, residue 490, residue 497, residue 2092, residue 2093 or two or more combinations thereof of full-length mature FVIII. In a particular embodiment, the amino acid substitution at residue 471, 484, or 497 is an amino acid other than arginine, the amino acid substitution at residue 487 is an amino acid other than tyrosine, the amino acid substitution at residue 2092 is an amino acid other than lysine, or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine.

[0026] In some embodiments, the FVIII protein contains at least one amino acid substitution, which induces the FVIII protein to be more stable than a FVIII

- 11 2018203206 08 May 2018 protein without the substitution. Such substitutions can be located in the A2 domain and the A3 domain of the FVIII protein, e.g., at a residue corresponding to residue 664, residue 1826, residue 662, residue 1828, or two or more combinations thereof of full-length mature FVIII.

[0027] The VWF fragment useful for the present invention comprises a D' domain and D3 domain, which together are capable of binding to FVIII. The VWF fragment can comprise the amino acid sequence of the D' domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2 and/or the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2. In one embodiment, the VWF fragment is a monomer. In another embodiment, the VWF fragment comprises at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments. In one embodiment, the two or more VWF fragments may be identical or they may be different. The VWF fragment can comprise an amino acid at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2. The VWF fragment may consist essentially of or consist of amino acids 764 to 1240 of SEQ ID NO: 2. In certain embodiments, the VWF fragment can contain at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. In other embodiments, the VWF fragment further comprises the Dl domain, the D2 domain, or the Dl and D2 domains of VWF.

[0028] The VWF fragment may further comprise a VWF domain selected from the group consisting of the Al domain, the A2 domain, the A3 domain, the D4 domain, the BI domain, the B2 domain, the B3 domain, the Cl domain, the C2 domain, the CK domain, one or more fragments thereof, and any combinations thereof. For example, the VWF fragment can consist essentially of or consist of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al domains of VWF or fragments thereof.

- 122018203206 08 May 2018

In some embodiments, the VWF fragment further comprises a signal peptide of

VWF or FVIII which is operably linked to the VWF fragment.

[0029] One or more of the linkers useful in the invention have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids. In some embodiments, one or more of the linkers have a length of about 1 to about 2000 amino acids. In one embodiment, one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids. In another embodiment, one or more of the linkers comprise a gly/ser peptide, an XTEN sequence, or both. Examples of the gly/ser peptide include, but are not limited to, a formula of (Gly4Ser)_n (SEQ ID NO: 139) or S(Gly4Ser)_n (SEQ ID NO: 140), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. For example, the (Gly4Ser)_n linker can be (Gly₄Ser)₃ (SEQ ID NO: 63) or (Gly₄Ser)₄ (SEQ ID NO: 138). In one embodiment, the linker comprises at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both. In another embodiment, the linker comprises 20 amino acids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acids thrombin cleavable linker. The cleavable linkers can comprise one or more of the cleavage sites by a protease selected from the group consisting of factor XIa, factor Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase-2, Granzyme-B, TEV, Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP-20, e.g., TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8). Nonlimiting examples of one or more of the cleavage sites comprise an amino acid sequence selected from the group consisting of RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO:23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27),

- 13 2018203206 08 May 2018

GPAGLGGA (SEQ ID NO: 28), GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34), and LPKTGSES (SEQ ID NO: 35). In some embodiments, the first cleavage site and the second cleavage site are identical or different.

[0030] The XTEN sequence useful for the invention can be selected from the group consisting of AE42 (SEQ ID NO: 36), AE144 (SEQ ID NO: 37), AG144 (SEQ ID NO: 38), AE288 (SEQ ID NO: 39), AG288 (SEQ ID NO: 40), AE576 (SEQ ID NO: 41). AG576 (SEQ ID NO: 42), AE864 (SEQ ID NO: 43), AE72 (SEQ ID NO: 127), AE144 2A (SEQ ID NO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ ID NO: 131), AE144 6B (SEQ ID NO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ ID NO: 134), AG144C (SEQ ID NO: 135), AG144 F (SEQ ID NO: 136), AE288 2 (SEQ ID NO: 137), or AG864 (SEQ ID NO: 44). In a particular embodiment, the XTEN sequence comprises AE288 or AG288.

[0031] The chimeric protein of the invention can be polysialylated, pegylated, or hesylated.

[0032] The present invention is also directed to a polynucleotide or a set of polynucleotides encoding the chimeric protein. The polynucleotide can further comprise a polynucleotide chain, which encodes PC5 or PC7. The invention is also directed to a vector comprising the polynucleotide or the set of polynucleotides and one or more promoter operably linked to the polynucleotide or the set of polynucleotides. The vector can further comprise an additional vector, which comprises a polynucleotide chain encoding PC5 or PC7. The invention is also drawn to a host cell comprising the polynucleotide or the vector. The host cell can be a mammalian cell, e.g., HEK293 cell, CHO cell, or BHK cell. In some embodiments, the PC5 or PC7 of the host cell cleaves the D1D2 domains of VWF.

[0033] The invention is also directed to a pharmaceutical composition comprising the chimeric protein, the polynucleotide, the vector, or the host cell, and a pharmaceutically acceptable carrier. The composition of the invention thus has an extended half-life compared to wild type FVIII protein. The half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about

- 142018203206 08 May 2018

2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. The half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

[0034] The composition of the present invention can be administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration. In one embodiment, the composition is administered via parenteral administration, e.g., intravenous or subcutaneous administration. The composition of the invention is useful to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof. In one embodiment, the subject treated with the chimeric protein is scheduled to undergo a surgery. In another embodiment, the treatment is prophylactic or on-demand.

[0035] The invention is also directed to a method of preventing or inhibiting binding of a FVIII protein with endogenous VWF comprising adding an effective amount of the chimeric protein, the polynucleotide vector, the host cell, or the composition to a subject in need thereof, wherein the VWF fragment binds to the

FVIII protein and thus prevents or inhibits binding of endogenous VWF. The

- 15 2018203206 08 May 2018 present invention is further directed to a method of extending or increasing the half-life of the FVIII protein, wherein the method comprises administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases the half-life of the FVIII protein. Also provided is a method of preventing or inhibiting clearance of a FVIII protein from a cell, wherein the method comprises administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition to a cell comprising a FVIII protein or a polynucleotide encoding the FVIII protein, wherein the protein having VWF activity binds to the FVIII protein. The subject useful for the present methods is an animal, e.g., a human, e.g., a patient suffering from hemophilia A.

[0036] The present invention also provides a method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein, the polynucleotide, the vector, the host cell, or the composition, wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath. The treatment can be prophylactic or on-demand. In one embodiment, the effective amount is 0.1 pg/kg to 500 mg/kg.

[0037] The invention also includes a method of making a chimeric protein, comprising transfecting one or more host cell with the polynucleotide or the vector and expressing the chimeric protein in the host cell.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES [0038] Figure 1A-D. Schematic diagrams of VWF fragments. Fig. 1A shows three exemplary VWF fragments useful for the invention, e.g., VWF-002, VWF010, and VWF-013. VWF-002 contains amino acids 1 to 477 of SEQ ID NO: 124 (amino acids 764 to 1240 of SEQ ID NO: 2) and is synthesized without the pre/propeptide sequences. VWF-010 contains the D1D2 domains in addition to the D'D3 domains. VWF-013 contains the D1D2DD3 domains in addition to

- 162018203206 08 May 2018 alanine residues substituting cysteines at residues 336 and 379 of SEQ ID NO:

123 Fig. IB shows VWF-031, which contains the D1D2D'D3 domains fused to an Ig constant region or a portion thereof, e.g., an Fc region, by a cleavable linker, e.g., a 48 amino acids thrombin cleavable linker. Fig. 1C shows VWF-025, which is a nucleotide sequence encoding D1D2D'D3 domains contained in pLIVE vector, and VWF-029, which is a nucleotide sequence encoding D1D2D'D3 domains with two amino acid substitutions, C336A and C379A, in pLIVE vector. Fig. ID shows full-length VWF fragment comprising propeptide (the DI and D2 domains) and mature subunits (the D', D3, Al, A2, A3, D4, Bl-3, Cl-2 domains). The VWF fragment is about 250 kDa protein and forms multimers (> 20 MDa) by disulfide bonding. The VWF fragment associates with FVIII (95-98%) in noncovalent complex and then extends half-life of FVIII by protecting FVIII from protease cleavage/activation, stabilizing heavy & light chain, and preventing clearance of FVIII by scavenger receptors. The VWF fragment also can limit half-life of FVIII by clearance of FVIII-VWF complex through VWF receptors and preventing pinocytosis and recycling of rFVIIIFc.

[0039] Figure 2. Pharmacokinetic profile of rFVIII-XTEN (rFVIII-AE288 or rFVIII-288AE) in VWF D’D3 expression mice or in FVIII and VWF double knockout (DKO) mice. Figure 2A shows the timeline of hydrodynamic injection (HDI) of the DD3 domain encoding plasmid DNA (VWF-025) (day -5), intravenous dosing of rFVIII-XTEN AE288 (day 0), and PK sample collection (day 5). Figure 2B shows FVIII activity measured by a FVIII chromogenic assay after IV dosing of rFVIII-XTEN288 in D1D2DO3 mice (inverted triangle) and rFVIII-XTEN288 in DKO mice (diamond). Fig. 2C shows the DD3 plasma level (ng/mL) after administration of VWF-025. The X axis represents time in hours.

[0040] Figure 3. Schematic diagram of exemplary VWF:FVIII heterodimer constructs. The constructs have the common structure represented as formula FVIII-F1-L1-V-X-L2-F2, but contain examples of different variable linkers. The construct (FVIII-161) shown contains a heterodimeric FVIII (the heavy chain and the light chain are associated by a metal bond) linked to a first Fc region and a VWF fragment, which is the D’ and D3 domains of VWF (i.e., amino acids 1 to 477 of SEQ ID NO: 2 with amino acid substitutions C336A and C379A) linked to an XTEN sequence, which is further linked to a cleavable linker and a second Fc

- 172018203206 08 May 2018 region. The XTEN sequence contained in FVIII-161 is an ΧΤΕΝ AE288 sequence, and the linker is a thrombin cleavable linker, which has 35 amino acids. In FVIII-161, the FVIII protein linked to the first Fc region is linked to the VWF fragment by a processable linker. Upon expression, the processable linker can be cleaved by an intracellular processing enzyme, thus making the construct three polypeptide chains associated with each other.

[0041] Figure 4 is schematic diagrams of FVIII-VWF heterodimer or monomer examples. FVIII-168, FVIII-175, FVIII-172, FVIII-174, and FVIII170. Construct FVIII-168 comprises a single chain FVIII sequence (having an alanine residue substitute the arginine residues at residues 1645 and 1648) linked to a first Fc region, which is then fused to a VWF fragment linked to a second Fc region by a thrombin cleavable linker, which has 48 amino acids. AE288 XTEN is inserted in the B domain of the single chain FVIII sequence. The linkage between the first Fc region and the VWF fragment comprises a linker that is capable of being cleaved by an intracellular processing enzyme, i.e., processable linker. Construct FVIII175 comprises a single chain FVIII (having an alanine residue substitute the arginine residues at residues 1645 and 1648) linked to AE288 XTEN and a first Fc region, which is linked to a second Fc region by a linker, e.g., a processable linker. AE288 XTEN is inserted in the B domain of the single chain FVIII sequence. Construct FVIII-172 comprises two polypeptide chains, a first chain comprising a heavy chain FVIII sequence fused to AE288 XTEN, a second chain comprising a light chain FVIII sequence, a first Fc region, a linker (e.g., a processable linker), a VWF fragment, a thrombin cleavable linker (e.g., 48 amino acids), and a second Fc region. Construct FVIII-174 comprises two polypeptide chains, a first chain comprising a heavy chain FVIII sequence fused to AE288 XTEN and a second chain comprises a light chain FVIII, a first Fc region, a linker (e.g., a processable linker), and a second Fc region. Construct FVIII-170 comprises a VWF fragment, AE288 XTEN, a linker (e.g., a thrombin cleavable linker, which is 35 amino acids in length), and a single chain FVIII sequence.

[0042] Figure 5. Pharmacokinetic profile of FVIII/VWF heterodimers containing an XTEN sequence in combination with an Fc region. Constructs FVIII-161,

FVIII-168, and FVIII-172 were administered to FVIII:VWF double knockout (DKO) mice by Hydrodynamic injection (HDI) at lOOug/mouse dose. Construct

- 182018203206 08 May 2018

FVIII-170 was administered to FVIITVWF DKO mice by HDI at 50 pg/mousc dose. The post-HDI plasma FVIII activity was analyzed by FVIII chromogenic assay for 24 hr post-HDI. The FVIII activity of the FVIITVWF heterodimers containing an XTEN sequence and Fc domains was compared with the FVIII activity of BDD-FVIII without the VWF fragment, XTEN sequence, and Fc domains.

[0043] Figure 6. Schematic diagrams of FVIII-VWF heterodimer examples cotransfection system. Fig. 6A. Construct FVIII-169 contains the full-length FVIII sequence (with an alanine residue substituting the arginine residues at 1645 and 1648 and with an XTEN sequence inserted in the single chain FVIII sequence), which is linked to an Fc region. VWF-031 contains the D1D2D'D3 fragment (with an alanine residue substituting the cysteine residues at 336 and 379) which is linked to another Fc region with a 48 thrombin cleavable linker. After intracellular processing, construct FVIII-169 produces a full length single chain FVIII (SCFVIII) fused to one Fc fragment and an XTEN sequence, and construct VWF-031 produces a 477 amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds can be formed between the Fc fragments that are linked to the SC FVIII or the D'D3 fragment, this in turn allows a non-covalent association of FVIII and D'D3. Fig. 6B. Construct FVIII-173 contains a heterodimeric FVIII sequence, a heavy chain FVIII sequence linked to an XTEN sequence and a light chain FVIII sequence linked to an Fc region. VWF-031 is described above. After intracellular processing, construct FVIII-173 produces a heterodimeric protein, a heavy chain FVIII fused to an XTEN sequence, a light chain FVIII fused to one Fc fragment, and construct VWF-031 produces a 477 amino acid D'D3 fragment linked to another Fc fragment. Two covalent bonds can be formed between the Fc fragments that are linked to the light chain FVIII or the D'D3 fragment, this in turn allows a non-covalent association of FVIII and D'D3.

[0044] Figure 7. Binding Affinity of Exemplary FVIITVWF containing an XTEN sequence and Fc domains to immobilized hVWF in Octet assay. The binding affinity for FVIII-169/VWF-031 and FVIII-057 (rFVIIIFc) fused to immobilized hVWF was tested using biolayer interferometry based measurements (Octet assay). Figure 7A shows binding response in nanomoles of FVIII169 and FVIIIFc

- 192018203206 08 May 2018 drug substance (a positive control) to immobilized hVWF. Figure 7B shows binding response of human IgGl (a negative control) to immobilized human VWF.

[0045] Figure 8. Pharmacokinetic (PK) profile of FVIII-169 in HemA and

FVIII:VWF double knockout (DKO) mice. Figure 8A shows the PK profile of FVIII-169/VWF-031 and FVIIIFc in HemA mice. HemA mice were treated with a single intravenous dose of FVIII-169/VWF-031 at 200 IU/kg. Plasma samples collected from the mice were tested by FVIII chromogenic assay. Half-life of FVIII-169/VWF-031 was calculated using WinNonlin program. Figure 8B shows the PK profde of FVIII-169/VWF-031, FVIII-169/Fc, and FVIIIFc in FVIII/VWF DKO mice.

[0046] Figure 9. PK profile of FVIII-ΧΤΕΝ variants in DD3 expressing

FVIII/VWF DKO mice. Figure 9A shows comparison of the PK profile of the FVIII-XTEN variants, FVIII with one XTEN, FVIII with two XTENs, and FVIII with three XTENs. One, two, or three XTENs were inserted in various portions of FVIII including C-terminus and B-domain. CT indicates that an XTEN is linked to the C-terminus of FVIII. Insertion site B/CT indicates that one XTEN is inserted between amino acid residue 745 and amino acid residue 746 of the FVIII protein and another XTEN is linked to the C-terminus of the FVIII protein. The amino acid residue numbering corresponds to the SQ BDD FVIII protein sequence. Insertion site 1900/B/CT indicates that a first XTEN is inserted between amino acid residue 1900 and amino acid residue 1901 of FVIII, a second XTEN is inserted between amino acid residue 745 and amino acid residue 746 of FVIII, and a third XTEN is linked to the C-terminus of FVIII. The mouse strain used to administer the FVIII-ΧΤΕΝ variants is a DKO mouse strain expressing D'D3 domains. Figure 9B shows the PK profile of FVIII-XTEN with three XTEN insertions. The FVIII-XTEN (1900/B/CT) variant was administered to either the FVIII/VWF DKO mice or HemA mice. The half-life of FVIII-XTEN (1900/B/CT) is compared.

[0047] Figure 10. FVIII activity of FVIIIFc (hollow triangle), FVIII169:Fc (filled circle), and FVIII 169 :VWF31 (hollow triangle) in mouse DKO plasma measured by chromogenic assay. FVIII:Fc contains a dual-chain FVIII (Heavy chain and

Light chain) fused to an Fc dimer (i.e., monomer-dimer hybrid). FVIII169 is

-202018203206 08 May 2018 described above (containing AE288 in the B domain, immediately downstream of amino acid 745 corresponding to mature FVIII sequence). FVIII169:Fc contains FVIII 169 fused to an Fc dimer. FVIII 169 :VWF31 contains VWF31 in addition to the Fc dimer, FVIII 169 fused to the first Fc region and VWF31 fused to the second Fc region, wherein the first Fc region and the second Fc region form a covalent bond, e.g., one or more disulfide bonds.

[0048] Figure 11. Effects of Fc, XTEN, and VWF-D'D3 fragments on FVIII halflife extension. BDD-FVIII (REFACTO®) (square), FVIIFc (circle), FVIII169/Fc (triangle), and FVIII 169/VWF031 (inverted triangle) were administered to FVIII and VWF double knockout (DKO) mice. The FVIII activity was measured by chromogenic assay, and the half-life was calculated using the WinNonlin-Phoenix program. X-axis shows time, and the Y-axis shows the FVIII plasma activity in mU/mL.

[0049] Figure 12A-C. Effects of different XTENs in rFVIII-XTEN/VWF heterodimer in HemA mice. Figure 12A shows the FVIII plasma activity normalized to 5 min value (%) of two XTENs inserted immediately downstream of residues 1900 and 1656 corresponding to mature FVIII sequence (i.e., FVIII195 (dual chain FVIII isoform) and FVIII-199 (single chain FVIII isoform)), compared to FVIII-169 containing an XTEN immediately downstream of residue 745 corresponding to mature FVIII sequence. FVIII-169/VWF-031 (filled circle), FVIII- 199/VWF-031 (filled square), and FVIII-195/VWF031 (hollow square) were administered in HemA mice to measure the FVIII plasma activity. Figure 12B shows the half-life extension effect of the second XTEN insertion immediately downstream of residues 403 (A2 domain) and 745 (B domain) (i.e., FVIII-203) and residues 745 (B domain) and 1900 (A3 domain) (FVIII-204) corresponding to mature FVIII sequence compared to FVIII-169 (an XTEN insertion in B domain only). FVIII-204/VWF031 (filled triangle), FVIII169/VWF-031 (filled circle), FVIII-203/VWF-031 (filled square), and scBDDFVIII (hollow diamond) were administered to HemA mice. The X-axis shows FVIII plasma activity normalized to 5 min value (%), and the y-axis shows time in hours. Figure 12C shows the half-life extension effect of the two XTEN insertions immediately downstream of residues 18 (Al domain) and 745 (B domain) (i.e., FVIII-205) compared to FVIII-169 (a single XTEN insertion in the B domain) and

-21 2018203206 08 May 2018 single chain FVIII without any Fc regions or any XTENs (i.e., FVIII-207). Figure 12C additionally shows the half-life extension effect of three XTEN insertions incorporated immediately downstream of residues 26 (Al domain), 1656 (A3 domain), and 1900 (A3 domain) (i.e., FVIII-201) compared to FVIII-169 (a single XTEN insertion immediately downstream of residue 745). . FVIII-205/VWF-031 (filled square), FVIII-201/VWF-031 (inverted triangle), FVIII-169/VWF-031 (filled circle), and FVIII-207 (hollow diamond) were administered to HemA mice. The FVIII plasma activity normalized to 5 min value (%) (X-axis) was measured over time in hours (Y-axis).

[0050] Figure 13. FVIII activity of rFVIII-XTEN/VWF-XTEN heterodimer in

FVIII/VWF DKO mice. FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity (X-axis) as a function of time (Y-axis) was plotted. FVIII-155 (scFVIIIFc without any XTENs) was co-expressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residue thrombin cleavable linker). The half-life of FVIII-155/VWF-034 was compared with that of FVIII-169/VWF-031, which has a AE 288 XTEN inserted into the B domain junction (immediately downstream of residue 745 corresponding to mature FVIII polypeptide) of FVIII.

[0051] Figure 14A-H. Schematic diagrams of various rFVIII-XTEN/VWF constructs. These constructs are also described in other sections herein. Fig. 14A shows single chain B domain deleted FVIII protein (sometimes indicated herein as scBDD-FVIII). The scBDD-FVIII constructs contain two substitutions at residues 1645 and 1648 from Arg to Ala. Figure 14B shows two polypeptide chain construct (FVIII155/VWF031), the first chain comprising single chain FVIII linked to an Fc region without any XTENS and the second chain comprising the VWF D'D3 fragment linked to an Fc region. This construct is used as a control. Fig. 14C shows two polypeptide chain construct (FVIII 199/VWF031), the first chain comprising single chain FVIII linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 1900 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 1656 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14D shows two polypeptide chain construct (FVIII201/VWF031), the first chain comprising single

-222018203206 08 May 2018 chain FVIII protein linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 26 corresponding to mature FVIII sequence, a second XTEN is inserted immediately downstream of residue 1656 corresponding to mature FVIII sequence, and a third XTEN is inserted immediately downstream of residue 1900 corresponding to mature FVIII sequence ,and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14E shows two polypeptide chain constructs (FVIII 169/VWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which an XTEN is inserted immediately downstream of residue 745 (indicated as B) corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14F shows two polypeptide chain construct (FVIII203/VWF031), the first chain comprising single chain FVIII protein, in which a first XTEN is inserted at residue 745 (B) corresponding to mature FVIII sequence and a second XTEN is inserted at residue 1900 corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14G shows two polypeptide chain construct (FVIII204/VWF031), the first chain comprising single chain FVIII protein linked to an Fc region, in which a first XTEN is inserted immediately downstream of residue 403 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 745 (B) corresponding to mature FVIII sequence, and a second chain comprising the VWF D'D3 fragment linked to an Fc region. Fig. 14H shows two polypeptide chain construct (FVIII205/VWF031), the first chain comprising single chain FVIII, in which a first XTEN is inserted immediately downstream of residue 18 corresponding to mature FVIII sequence and a second XTEN is inserted immediately downstream of residue 745 (B) corresponding to mature FVIII sequence, and the second chain comprising the VWF D'D3 fragment linked to an Fc region.

[0052] Figure 15. FVIII activity of rFVIII-XTEN/VWF and BDD-FVIII in

FVIII/VWF DKO mice. FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity (X-axis) as a function of time (Y-axis) was plotted. The half-life of rFVIII-XTEN/VWF (FVIII-205/VWF-031) was compared with that of BDD-FVIII and rFVIIIFc.

-23 2018203206 08 May 2018 [0053] Figure 16. Efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimers in HemA mice using tail clip bleeding model. The HemA mice tail clip bleeding model was used to compare the efficacy of FVIII169/VWF034, FVIII205/VWF031, and BDD-FVIII. The median blood loss in ml for 200 IU/kg of FVIII 169/VWF034 and FVIII205/VWF031 is compared with 200 IU/kg of BDD-FVIII, 65 IU/kg of BDD-FVIII, 20 IU/kg of BDD-FVIII, and vehicle.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS [0054] It is to be noted that the term a or an entity refers to one or more of that entity; for example, a nucleotide sequence, is understood to represent one or more nucleotide sequences. As such, the terms a (or an), one or more, and at least one can be used interchangeably herein.

[0055] The term polynucleotide or nucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide comprises a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term nucleic acid refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

-242018203206 08 May 2018 [0056] As used herein, a coding region or coding sequence is a portion of polynucleotide which consists of codons translatable into amino acids. Although a stop codon (TAG, TGA, or TAA) is typically not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode a binding domain-A and a binding domain-B as described below. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding domain of the invention. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0057] Certain proteins secreted by mammalian cells are associated with a secretory signal peptide which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide, and are cleaved from the complete or full-length polypeptide to produce a secreted or mature form of the polypeptide. In certain embodiments, a native signal peptide or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, e.g., a human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

[0058] The term downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide

-25 2018203206 08 May 2018 sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0059] The term upstream refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0060] As used herein, the term regulatory region refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0061] A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are operably associated if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

-262018203206 08 May 2018 [0062] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[0063] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picomaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0064] The term expression as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a gene product. As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0065] A vector refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. A replicon refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo,

-272018203206 08 May 2018

i.e., capable of replication under its own control. The term vector includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0066] Vectors may be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers may also be considered to be reporters.

[0067] The term plasmid refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or doublestranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0068] Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors.

Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

-282018203206 08 May 2018 [0069] A cloning vector refers to a replicon, which is a unit length of a nucleic acid that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria and expression in another, e.g., eukaryotic cells. Cloning vectors typically comprise one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

[0070] The term expression vector refers to a vehicle designed to enable the expression of an inserted nucleic acid sequence following insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with regulatory regions as described above.

[0071] Vectors are introduced into host cells by methods well known in the art,

e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter.

[0072] Culture, to culture and culturing, as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. Cultured cells, as used herein, means cells that are propagated in vitro.

[0073] As used herein, the term polypeptide is intended to encompass a singular polypeptide as well as plural polypeptides, and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, protein, amino acid chain, or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of polypeptide, and the term polypeptide can be used instead of, or interchangeably with any of these terms. The term polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups,

-292018203206 08 May 2018 proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

[0074] An isolated polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0075] Also included in the present invention are fragments or variants of polypeptides, and any combination thereof. The term fragment or variant when referring to polypeptide binding domains or binding molecules of the present invention include any polypeptides which retain at least some of the properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for an FVIII variant, or FVIII binding activity for the VWF fragment) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein, but do not include the naturally occurring full-length polypeptide (or mature polypeptide). Variants of polypeptide binding domains or binding molecules of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

[0076] The term VWF fragment or VWF fragments used herein means any

VWF fragments that interact with FVIII and retain at least one or more properties that are normally provided to FVIII by full-length VWF, e.g., preventing

-302018203206 08 May 2018 premature activation to FVIIIa, preventing premature proteolysis, preventing association with phospholipid membranes that could lead to premature clearance, preventing binding to FVIII clearance receptors that can bind naked FVIII but not VWF-bound FVIII, and/or stabilizing the FVIII heavy chain and light chain interactions.

[0077] A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

[0078] As known in the art, sequence identity between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive,

Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is

-31 2018203206 08 May 2018 calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0079] As used herein, an amino acid corresponding to or an equivalent amino acid in a VWF sequence or a FVIII protein sequence is identified by alignment to maximize the identity or similarity between a first VWF or FVIII sequence and a second VWF or FVIII sequence. The number used to identify an equivalent amino acid in a second VWF or FVIII sequence is based on the number used to identify the corresponding amino acid in the first VWF or FVIII sequence.

[0080] As used herein, the term insertion site refers to a position in a FVIII polypeptide, or fragment, variant, or derivative thereof, which is immediately upstream of the position at which a heterologous moiety can be inserted. An insertion site is specified as a number, the number being the number of the amino acid in mature native FVIII (SEQ ID NO:4) to which the insertion site corresponds, which is immediately N-terminal to the position of the insertion. For example, the phrase a3 comprises an XTEN at an insertion site which corresponds to amino acid 1656 of SEQ ID NO: 4 indicates that the heterologous moiety is located between two amino acids corresponding to amino acid 1656 and amino acid 1657 of SEQ ID NO: 4.

[0081] The phrase immediately downstream of an amino acid as used herein refers to position right next to the terminal carboxyl group of the amino acid. Similarly, the phrase immediately upstream of an amino acid refers to the position right next to the terminal amine group of the amino acid. Therefore, the phrase between two amino acids of an insertion site as used herein refers to a position in which an XTEN or any other polypeptide is inserted between two adjacent amino acids. Thus, the phrases inserted immediately downstream of an amino acid and inserted between two amino acids of an insertion site are used synonymously with inserted at an insertion site.

[0082] The terms inserted, is inserted, inserted into or grammatically related terms, as used herein refers to the position of an XTEN in a chimeric polypeptide relative to the analogous position in native mature human FVIII. As used herein the terms refer to the characteristics of the recombinant FVIII polypeptide relative to native mature human FVIII, and do not indicate, imply or infer any methods or

-322018203206 08 May 2018 process by which the chimeric polypeptide was made. For example, in reference to a chimeric polypeptide provided herein, the phrase an XTEN is inserted into immediately downstream of residue 745 of the FVIII polypeptide means that the chimeric polypeptide comprises an XTEN immediately downstream of an amino acid which corresponds to amino acid 745 in native mature human FVIII, e.g., bounded by amino acids corresponding to amino acids 745 and 746 of native mature human FVIII.

[0083] A fusion or chimeric protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, e.g., fusion of a Factor VIII domain of the invention with an lg Fc domain. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. A chimeric protein can further comprises a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non-covalent bond.

[0084] As used herein, the term half-life refers to a biological half-life of a particular polypeptide in vivo. Half-life may be represented by the time required for half the quantity administered to a subject to be cleared from the circulation and/or other tissues in the animal. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid aphase and longer β-phase. The α-phase typically represents an equilibration of the administered Fc polypeptide between the intra- and extra-vascular space and is, in part, determined by the size of the polypeptide. The β-phase typically represents the catabolism of the polypeptide in the intravascular space. In some embodiments, FVIII and chimeric proteins comprising FVIII are monophasic, and thus do not have an alpha phase, but just the single beta phase. Therefore, in certain embodiments, the term half-life as used herein refers to the half-life of the polypeptide in the β-phase. The typical β-phase half-life of a human antibody in humans is 21 days.

-33 2018203206 08 May 2018 [0085] The term linked as used herein refers to a first amino acid sequence or nucleotide sequence covalently or non-covalently joined to a second amino acid sequence or nucleotide sequence, respectively. The first amino acid or nucleotide sequence can be directly joined or juxtaposed to the second amino acid or nucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. The term linked means not only a fusion of a first amino acid sequence to a second amino acid sequence at the Cterminus or the N-terminus, but also includes insertion of the whole first amino acid sequence (or the second amino acid sequence) into any two amino acids in the second amino acid sequence (or the first amino acid sequence, respectively).

In one embodiment, the first amino acid sequence can be linked to a second amino acid sequence by a peptide bond or a linker. The first nucleotide sequence can be linked to a second nucleotide sequence by a phosphodiester bond or a linker. The linker can be a peptide or a polypeptide (for polypeptide chains) or a nucleotide or a nucleotide chain (for nucleotide chains) or any chemical moiety (for both polypeptide and polynucleotide chains). The term linked is also indicated by a hyphen (-).

[0086] As used herein the term associated with refers to a covalent or noncovalent bond formed between a first amino acid chain and a second amino acid chain. In one embodiment, the term associated with means a covalent, nonpeptide bond or a non-covalent bond. This association can be indicated by a colon, i.e., (:). In another embodiment, it means a covalent bond except a peptide bond. For example, the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CHI and CL regions are associated by a disulfide bond and the two heavy chains are associated by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system). Examples of covalent bonds include, but are not limited to, a peptide bond, a metal bond, a hydrogen bond, a disulfide bond, a sigma bond, a pi bond, a delta bond, a glycosidic bond, an agnostic bond, a bent bond, a dipolar bond, a Pi backbond, a double bond, a triple bond, a quadruple bond, a quintuple bond, a sextuple bond, conjugation, hyperconjugation, aromaticity, hapticity, or antibonding. Non-limiting examples

-342018203206 08 May 2018 of non-covalent bond include an ionic bond (e.g., cation-pi bond or salt bond), a metal bond, an hydrogen bond (e.g., dihydrogen bond, dihydrogen complex, lowbarrier hydrogen bond, or symmetric hydrogen bond), van der Walls force,

London dispersion force, a mechanical bond, a halogen bond, aurophilicity, intercalation, stacking, entropic force, or chemical polarity.

[0087] The term monomer-dimer hybrid used herein refers to a chimeric protein comprising a first polypeptide chain and a second polypeptide chain, which are associated with each other by a disulfide bond, wherein the first chain comprises a clotting factor, e.g., Factor VIII, and a first Fc region and the second chain comprises, consists essentially of, or consists of a second Fc region without the clotting factor. The monomer-dimer hybrid construct thus is a hybrid comprising a monomer aspect having only one clotting factor and a dimer aspect having two Fc regions.

[0088] As used herein, the term cleavage site or enzymatic cleavage site refers to a site recognized by an enzyme. Certain enzymatic cleavage sites comprise an intracellular processing site. In one embodiment, a polypeptide has an enzymatic cleavage site cleaved by an enzyme that is activated during the clotting cascade, such that cleavage of such sites occurs at the site of clot formation. Exemplary such sites include, e.g., those recognized by thrombin, Factor XIa or Factor Xa. Exemplary FXIa cleavage sites include, e.g., TQSFNDFTR (SEQ ID NO: 45) and SVSQTSKLTR (SEQ ID NO: 46). Exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 47), TTKIKPR (SEQ ID NO: 48), LVPRG (SEQ ID NO: 49) and ALRPR (amino acids 1 to 5 of SEQ ID NO: 50). Other enzymatic cleavage sites are known in the art.

[0089] As used herein, the term processing site or intracellular processing site refers to a type of enzymatic cleavage site in a polypeptide which is a target for enzymes that function after translation of the polypeptide. In one embodiment, such enzymes function during transport from the Golgi lumen to the trans-Golgi compartment. Intracellular processing enzymes cleave polypeptides prior to secretion of the protein from the cell. Examples of such processing sites include, e.g., those targeted by the PACE/furin (where PACE is an acronym for Paired basic Amino acid Cleaving Enzyme) family of endopeptidases. These enzymes are localized to the Golgi membrane and cleave proteins on the carboxyterminal

-35 2018203206 08 May 2018 side of the sequence motif Arg-[any residue]-(Lys or Arg)-Arg. As used herein the furin family of enzymes includes, e.g., PCSK1 (also known as PCl/Pc3), PCSK2 (also known as PC2), PCSK3 (also known as furin or PACE), PCSK4 (also known as PC4), PCSK5 (also known as PC5 or PC6), PCSK6 (also known as PACE4), or PCSK7 (also known as PC7/LPC, PC8, or SPC7). Other processing sites are known in the art.

[0090] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0091] The term Furin refers to the enzymes corresponding to EC No. 3.4.21.75.

Furin is subtilisin-like proprotein convertase, which is also known as PACE (Paired basic Amino acid Cleaving Enzyme). Furin deletes sections of inactive precursor proteins to convert them into biologically active proteins. During its intracellular transport, pro-peptide of VWF can be cleaved from mature VWF molecule by a Furin enzyme. In some embodiments, Furin cleaves the D1D2 from the D'D3 of VWF. In other embodiments, a nucleotide sequence encoding Furin can be expressed together with the nucleotide sequence encoding a VWF fragment so that D1D2 domains can be cleaved off intracellularly by Furin.

[0092] In constructs that include more than one processing or cleavage site, it will be understood that such sites may be the same or different.

[0093] A processable linker as used herein refers to a linker comprising at least one intracellular processing site, which is described elsewhere herein.

[0094] Hemostatic disorder, as used herein, means a genetically inherited or acquired condition characterized by a tendency to hemorrhage, either spontaneously or as a result of trauma, due to an impaired ability or inability to form a fibrin clot. Examples of such disorders include the hemophilias. The three main forms are hemophilia A (factor VIII deficiency), hemophilia B (factor IX deficiency or Christmas disease) and hemophilia C (factor XI deficiency, mild bleeding tendency). Other hemostatic disorders include, e.g., Von Willebrand disease, Factor XI deficiency (PTA deficiency), Factor XII deficiency, deficiencies or structural abnormalities in fibrinogen, prothrombin, Factor V, Factor VII, Factor X or factor XIII, Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb, the receptor for VWF, can be defective and lead to lack of primary clot formation (primary hemostasis) and increased bleeding

-362018203206 08 May 2018 tendency), and thrombasthenia of Glanzman and Naegeli (Glanzmann thrombasthenia). In liver failure (acute and chronic forms), there is insufficient production of coagulation factors by the liver; this may increase bleeding risk.

[0095] The chimeric molecules of the invention can be used prophylactically. As used herein the term prophylactic treatment refers to the administration of a molecule prior to a bleeding episode. In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to or after surgery as a prophylactic. The chimeric protein of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, dental procedures, or stem cell transplantation.

[0096] The chimeric protein of the invention is also used for on-demand treatment. The term on-demand treatment refers to the administration of a chimeric molecule in response to symptoms of a bleeding episode or before an activity that may cause bleeding. In one aspect, the on-demand treatment can be given to a subject when bleeding starts, such as after an injury, or when bleeding is expected, such as before surgery. In another aspect, the on-demand treatment can be given prior to activities that increase the risk of bleeding, such as contact sports.

[0097] As used herein the term acute bleeding refers to a bleeding episode regardless of the underlying cause. For example, a subject may have trauma, uremia, a hereditary bleeding disorder (e.g., factor VII deficiency) a platelet disorder, or resistance owing to the development of antibodies to clotting factors.

[0098] Treat, treatment, treating, as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition, or the prophylaxis of one or more symptoms associated with a disease or condition. In one embodiment, the term treating or treatment means maintaining a FVIII trough level at least about 1 IU/dL, 2 IU/dL, 3 IU/dL, 4 IU/dL, 5 IU/dL, 6 IU/dL, 7 IU/dL, 8 IU/dL, 9 IU/dL,

IU/dL, 11 IU/dL, 12 IU/dL, 13 IU/dL, 14 IU/dL, 15 IU/dL, 16 IU/dL, 17

-372018203206 08 May 2018

IU/dL, 18 IU/dL, 19 IU/dL, or 20 IU/dL in a subject by administering a chimeric protein or a VWF fragment of the invention. In another embodiment, treating or treatment means maintaining a FVIII trough level between about 1 and about 20 IU/dL, about 2 and about 20 IU/dL, about 3 and about 20 IU/dL, about 4 and about 20 IU/dL, about 5 and about 20 IU/dL, about 6 and about 20 IU/dL, about 7 and about 20 IU/dL, about 8 and about 20 IU/dL, about 9 and about 20 IU/dL, or about 10 and about 20 IU/dL. Treatment or treating of a disease or condition can also include maintaining FVIII activity in a subject at a level comparable to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the FVIII activity in a non-hemophiliac subject. The minimum trough level required for treatment can be measured by one or more known methods and can be adjusted (increased or decreased) for each person.

Chimeric Proteins [0099] The present invention is directed to extending the half-life of a Factor VIII protein using a VWF fragment and an XTEN sequence by preventing or inhibiting a FVIII half-life limiting factor, i.e., endogenous VWF, from associating with the FVIII protein. Endogenous VWF associates with about 95% to about 98% of FVIII in non-covalent complexes. While endogenous VWF is a FVIII half-life limiting factor, endogenous VWF bound to a FVIII protein is also known to protect FVIII in various ways. For example, full length VWF (as a multimer having about 250 kDa) can protect FVIII from protease cleavage and FVIII activation, stabilize the FVIII heavy chain and/or light chain, and prevent clearance of FVIII by scavenger receptors. But, at the same time, endogenous VWF limits the FVIII half-life by preventing pinocytosis and by clearing FVIII VWF complex from the system through the VWF clearance pathway. It is believed, while not bound by a theory, that endogenous VWF is a half-life limiting factor that prevents the half-life of a FVIII protein fused to a half-life extender from being longer than about two-fold that of wild-type FVIII. Therefore, the present invention is directed to preventing or inhibiting interaction between endogenous VWF and a FVIII protein using a VWF fragment, thereby increasing a half-life of the FVIII protein by using an XTEN sequence alone or an XTEN sequence in combination with an Ig constant region or a portion thereof The

-382018203206 08 May 2018 [0100] [0101]

XTEN sequence can be linked to the FVIII protein or the VWF fragment. The FVIII protein associated with the VWF fragment is thus cleared from the circulation more slowly by one or more VWF clearance receptors and then can have the full half-life extension of the XTEN sequence or the XTEN sequence in combination of the Ig constant region, as compared to wild type FVIII or a FVIII protein without the VWF fragment.

In one embodiment, a VWF fragment is associated (or linked) with the FVIII protein by a covalent or a non-covalent bond. In some instances, however, the physical blockage or chemical association (e.g., non-covalent bonding) between the VWF fragment and the FVIII protein may not be strong enough to provide a stable complex comprising the FVIII protein and the VWF fragment in the presence of endogenous VWF. For example, a VWF fragment forming a noncovalent bond with a FVIII protein without any other connections may readily be dissociated in vivo from the FVIII protein in the presence of endogenous VWF, replacing the VWF fragment (e.g., recombinant VWF, i.e., rVWF) with endogenous VWF. Therefore, the FVIII protein non-covalently bound to endogenous VWF would undergo the VWF clearance pathway and be readily cleared from the system. In order to prevent the dissociation of the VWF fragment with the FVIII protein, in some embodiments, the association or linkage between the FVIII protein and the VWF fragment is a covalent bond, e.g., a peptide bond, one or more amino acids, or a disulfide bond. In certain embodiments, the association (i.e., linkage) between the adjunct moiety and the FVIII protein is a peptide bond or a linker between the FVIII protein and the VWF fragment (FVIII/VWF linker). Non-limiting examples of the linker are described elsewhere herein. In some embodiments, the VWF fragment is a polypeptide comprising, consisting essentially of, or consisting of at least about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 4000 amino acids. Nonlimiting examples of the VWF fragment are described elsewhere herein.

In certain embodiments, the VWF fragment chemically (e.g., noncovalently) binds to or physically blocks one or more VWF binding sites on a FVIII protein. The VWF binding site on a FVIII protein is located within the A3 domain or the C2 domain of the FVIII protein. In still other embodiments, the

-392018203206 08 May 2018 [0102] [0103] [0104]

VWF binding site on a FVIII protein is located within the A3 domain and C2 domain. For example, the VWF binding site on a FVIII protein can correspond to amino acids 1669 to 1689 and/or 2303 to 2332 of SEQ ID NO: 4 [full-length mature FVIII],

The invention also provides a chimeric protein (comprising a FVIII protein and a VWF fragment) further comprising one or more XTEN sequences, which provide additional half-life extension properties. The one or more XTEN sequences can be inserted within the FVIII protein or the VWF fragment or linked to the N-terminus or the C-terminus of the FVIII protein or the VWF fragment. The invention also includes a FVIII protein linked to an XTEN sequence (a first half-life extending moiety) and an Ig constant region or a portion thereof (a second half-life extending moiety) so that the two half-life extending moieties extend the half-life of the FVIII protein through two different mechanisms.

In some embodiments, a chimeric protein comprises a FVIII protein linked to a first Ig constant region or a portion thereof (e.g., a first FcRn binding partner), a VWF fragment linked to a second Ig constant region or a portion thereof (e.g., a second FcRn binding partner), and one or more XTEN sequences inserted or linked to the FVIII protein or the VWF fragment, wherein the VWF fragment prevents the FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein, wherein the first and second Ig constant regions or portions thereof forms a covalent bond, e.g., a disulfide bond, and the one or more XTEN sequences extends the half-life of the FVIII protein.

In certain embodiments, a chimeric protein of the invention comprises a FVIII protein linked to a VWF fragment by an optional linker (i.e., FVIII/VWF linker) and one or more XTEN sequences inserted or linked to the FVIII protein or the VWF fragment, wherein the VWF fragment prevents the FVIII half-life limiting factor (e.g., endogenous VWF) from binding to the FVIII protein and the one or more XTEN sequences extends the half-life of the FVIII protein. In one aspect, the optional linker (FVIII/VWF linker) comprises a sortase recognition motif. In another aspect, the optional linker (FVIII/VWF linker) comprises a cleavable site. Examples of the cleavage linker (i.e., linker containing one or more cleavage site) are described elsewhere herein.

-402018203206 08 May 2018 [0105] [0106]

The chimeric protein of the present invention includes, but is not limited to:

(1) a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, and FVIII, wherein the XTEN sequence is linked to the VWF fragment;

(2) a FVIII protein, an XTEN sequence, and an Ig constant region or a portion thereof, wherein the FVIII protein is linked to an XTEN sequence and the Ig constant region or a portion thereof, or (3) a FVIII protein, an XTEN sequence, and a VWF fragment, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or N-terminus or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) of FVIII, and the VWF fragment and the FVIII protein are associated with each other.

(1) Von Willebrand Factor (VWF) fragment linked to XTEN, and FVIII

The present invention is directed to a chimeric protein comprising (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein (i), (ii), and (iii) are linked to or associated with each other. The VWF fragment linked to the XTEN sequence, as a part of a chimeric protein in the present invention, associates with the FVIII protein, thus preventing or inhibiting interaction between endogenous VWF and the FVIII protein. In certain embodiments, the VWF fragment, which is capable of preventing or inhibiting binding of the FVIII protein with endogenous VWF, can at the same time have at least one VWF-like FVIII protecting property. Examples of the VWF-like FVIII protecting properties include, but are not limited to, protecting FVIII from protease cleavage and FVIII activation, stabilizing the FVIII heavy chain and/or light chain, and preventing clearance of FVIII by scavenger receptors. As a result, the VWF fragment can prevent clearance of the FVIII protein through the VWF clearance pathway, thus reducing clearance of FVIII from the circulatory system. In some embodiments, the VWF fragments of the present invention bind to or are associated with a FVIII protein and/or physically or chemically block the VWF binding site on the FVIII protein. The FVIII protein associated with the VWF fragment is thus cleared from the circulation more slowly, as compared to wild type FVIII or FVIII not associated with the VWF fragment.

-41 2018203206 08 May 2018 [0107] [0108]

In one embodiment, the invention is directed to a chimeric protein comprising (i) a VWF fragment comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the XTEN sequence is linked to the VWF fragment (e.g., (al) V-X or (a2) X-V, wherein V comprises a VWF fragment and X comprises an XTEN sequence), and the VWF fragment is linked to or associated with the FVIII protein. In another embodiment, the VWF fragment and the XTEN sequence can be linked by a linker (e.g., (a3) V-F-X or (a4) X-F-V) or a peptide bond. The linker can be a cleavable linker, e.g., a thrombin cleavable linker, which can be cleaved at the site of coagulation. In other embodiments, the VWF fragment, the XTEN sequence, and the FVIII protein are placed in a single polypeptide chain. In still other embodiments, the chimeric protein comprises two polypeptide chains, a first chain comprising the VWF fragment and the XTEN sequence and a second chain comprising the FVIII protein. In yet other embodiments, the chimeric protein comprises three polypeptide chains, a first chain comprising the VWF fragment and the XTEN sequence, a second chain comprising a light chain of FVIII and a third chain comprising a heavy chain of FVIII, wherein the first chain and the second chain are associated with each other (e.g., covalent bond, e.g., disulfide bond), and the second chain and the third chain are associated with each other (e.g., metal bond). In still other embodiments, the XTEN sequence can be linked to the N-terminus or the C-terminus of the VWF fragment or inserted immediately downstream of one or more amino acids in the VWF fragment.

In certain embodiments, a chimeric protein of the invention comprises a formula comprising:

(a) V-X-FVIII, (b) FVIII-X-V, (c) V-X:FVIII, (d) X-V:FVIII, (e) FVIII:V-X, (f) FVIII:X-V, or (a5) X-V-FVIII, wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences,

-422018203206 08 May 2018 [0109]

FVIII comprises a FVIII protein;

(-) represents a peptide bond or one or more amino acids; and (:) is a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a nonpeptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula V-X-FVIII means formula NH2-VX-FVIII -COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. For example, formula V-XFVIII can further comprise any sequences at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII unless otherwise specified. In another embodiment, the hyphen (-) indicates a peptide bond.

In other embodiments, a chimeric protein of the invention comprises a formula comprising:

(a) V(X1)-X2-FVIII, (b) FVIII-X2-V(X1), (c) V(X1):FVIII, (d) FVIII:V(X1), or (a5) X2-V(X1)-FVIII, wherein V(X1) comprises a VWF fragment and a first XTEN sequence (XI), wherein the XTEN sequence is inserted immediately downstream of one or more amino acids in the VWF fragment,

X2 comprises one or more optional XTEN sequences,

FVIII comprises a FVIII protein;

-43 2018203206 08 May 2018 [0110] (-) is a peptide bond or one or more amino acids; and (:) is a chemical association or a physical association.

In some embodiments, a chimeric protein comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) a first optional linker, and (v) a second optional linker, wherein the XTEN sequence is linked to the VWF fragment and/or to the FVIII protein by the linker. In certain embodiments, a chimeric protein comprises a formula

comprising:
(bl)	V-L1-X-L2-FVIII,
(b2)	FVIII-L2-X-L1-V,
(b3)	V-L1-X:FVIII,
(b4)	X-L1-V:FVIII,
(b5)	FVIII:V-L1-X,
(b6)	FVIII:X-L1-V,
(b7)	X-L1-V-L2-FVIII, or
(b8)	FVIII-L2-V-L1-X,

wherein V comprises a VWF fragment,

X comprises one or more XTEN sequences,

FVIII comprises a FVIII protein,

LI comprises a first optional linker, e.g., a first cleavable linker,

L2 comprises a second optional linker, e.g., a second cleavable linker or an optional processable linker;

(-) is a peptide bond or one or amino acids; and (:) is a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a nonpeptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence

-442018203206 08 May 2018 [0111] from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula (bl) V-L1-X-L2-FVIII means formula NH2-V-L1-X-L2-FVIII-COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. In another embodiment, the hyphen (-) indicates a peptide bond.

Another aspect of the present invention is to provide a FVIII chimeric protein having reduced or no interactions with a FVIII half-life limiting factor, e.g., endogenous VWF, and at the same time maximizing the half-life of the FVIII protein using an XTEN sequence (a first half-life extender) in combination with a second half-life extender or a moiety providing a covalent bond between the FVIII protein and the VWF fragment, e.g., an Ig constant region or a portion thereof. In one embodiment, a chimeric protein of the invention comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, and (iv) an Ig constant region or a portion thereof (also referred to herein as F), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker, (2) the VWF fragment is associated with or linked to the FVIII protein by an additional optional linker, e.g., a cleavable linker, and (3) the Ig constant region or a portion thereof is linked to the VWF fragment, the XTEN sequence, or the FVIII protein. In another embodiment, a chimeric protein of the invention comprises (i) a VWF fragment comprising a D' domain and a D3 domain of VWF, (ii) an XTEN sequence, (iii) a FVIII protein, (iv) an Ig constant region or a portion thereof (FI or a first Ig constant region or a portion thereof), and (v) an additional Ig constant region or a portion thereof (F2 or a second Ig constant region or a portion thereof), wherein (1) the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker, (2) the XTEN sequence or the VWF fragment is linked to the Ig constant region or a portion thereof, (3) the FVIII is linked to the additional Ig constant region or a portion thereof, and (4) the Ig constant region or a portion thereof is associated with or linked to the additional Ig constant region or a portion thereof. In one embodiment, the association or linkage between the two Ig constant regions or a portion thereof is a covalent bond, e.g., a disulfide bond. In

-45 2018203206 08 May 2018 [0112] [0113] another embodiment, the association or linkage between the two Ig constant regions or a portion thereof is a processable linker, wherein the processable linker is intracellularly processed by a protease. For example, the chimeric protein comprises a formula comprising:

(g) V-L2-X-L1-F1: FVIII-L3-F2;

(h) V-L2-X-L1-F1:F2-L3-FVIII;

(i) F-L1-X-L2-V: FVIII-L3-F2;

(j) F-L1-X-L2-V:F2-L3-FVIII;

(k) V-L2-X-L1-F1-L4-FVIII-L3-F2;

(l) F2-L3-FVIII-L4-F1-L1-X-L2-V;

(m) FVIII-L2-F2-L4-V-L2-X-L1-F1; or (n) F1-L1-X-L2-V-L4-F2-L2-FVIII, wherein V comprises a VWF fragment, each of LI and L3 comprises an optional linker,

L2 comprises an optional linker, e.g., a cleavable linker,

L4 is an optional linker, e.g., a processable linker,

FVIII comprises a FVIII protein,

X comprises one or more XTEN sequences,

FI comprises an optional Ig constant region or a portion thereof,

F2 comprises an optional additional Ig constant region or a portion thereof;

(-) is a peptide bond or one or more amino acids; and (:) is a chemical association or a physical association.

In some embodiments, the FVIII protein in any constructs or formulas disclosed herein can further comprises at least one, at least two, at least three, at least four, at least five, or at least six XTEN sequences, each of the XTEN sequences inserted immediately downstream of one or more amino acids in the FVIII protein or linked to the N-terminus or the C-terminus of the FVIII protein. Non-limiting examples of the XTEN insertion sites are disclosed elsewhere herein.

In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a noncovalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a

-462018203206 08 May 2018 [0114] hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula (η) ΓΙΕ 1-X-L2-V-L4-F2-L2-F VIII means formula NH2-F1-LI-X-L2-V-L4-F2-L2FVIII -COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. In another embodiment, the hyphen (-) indicates a peptide bond.

In one embodiment, either or both of the lg constant region or a portion thereof (sometimes indicated herein by F or FI) and the additional lg constant region or a portion thereof (sometimes indicated herein by F2) linked to the VWF fragment or the FVIII protein can extend the half-life of the VWF fragment, the FVIII protein, or both. In another embodiment, a pair of the lg constant region or a portion thereof (sometimes indicated herein by F or FI) and the additional lg constant region or a portion thereof (sometimes indicated herein by F2), each of which are linked to the VWF fragment and the FVIII protein, provides a bond stronger than the non-covalent bond between the FVIII protein and the VWF fragment, i.e., a covalent bond, e.g., a disulfide bond, thereby preventing endogenous VWF from replacing the VWF fragment in vivo. FI or F2 can comprise an Fc region or an FcRn binding partner. In other embodiments, either or both of FI and F2 linked to the VWF fragment and/or the FVIII protein form a covalent bond (e.g., a disulfide bond) between FI and F2, thereby placing the VWF fragment and the FVIII protein in close proximity to prevent interaction of the FVIII protein with the VWF fragment. In some embodiments, FI and F2 are identical or different. Non-limiting examples of FI and F2 can be selected from the group consisting of a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragments, derivatives, or analogs thereof, and two or more combinations thereof. In one embodiment, FI, F2, or both

-472018203206 08 May 2018 [0115] comprise at least one CHI domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or the functional fragments, derivatives, or analogs thereof. In another embodiment, FI, F2, or both comprise at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in the hinge-CH2 orientation). In other embodiments, FI, F2, or both comprise at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in the CH2-CH3 orientation.) Examples of the combination include, but are not limited to, a CH2 domain, a CH3 domain, and a hinge domain, which are also known as an Fc region (or Fc domain), e.g., a first Fc region or a first FcRn binding partner for Ff and a second Fc region or a second FcRn binding partner for F2. In other embodiments, Ff is linked to the VWF fragment by a linker, and/or F2 is linked to the FVIII protein by a linker. In some embodiments, FI and/or F2 comprises, consisting essentially of, or consisting of a hinge region. Additional non-limiting examples of the Fc regions or the FcRn binding partners are described elsewhere herein.

In certain embodiments, a chimeric protein of the invention comprises two polypeptide chains, a first polypeptide chain comprising, consisting essentially of, or consisting of a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, a first Ig constant region or a portion thereof (e.g., a first Fc region), and an optional linker between the VWF fragment and the XTEN sequence or the XTEN sequence or the first Ig constant region or a portion thereof and a second polypeptide chain comprising, consisting essentially of, or consisting of a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region). The linker between the VWF fragment and the first Ig constant region or a portion thereof can be a cleavable linker, e.g., a thrombin cleavable linker, which can be cleaved at the site of coagulation. In some embodiments, the first polypeptide chain and the second polypeptide chain are associated with each other. The association between the first chain and the second chain prevents replacement of the first chain comprising the VWF fragment with endogenous VWF in vivo. In one embodiment, the association between the first chain and the second chain can be a covalent bond. In a particular embodiment, the covalent bond is a disulfide bond. In some embodiments, the FVIII protein in the second chain further comprises one or more XTEN sequences linked to the C-terminus or

-482018203206 08 May 2018 [0116] [0117]

N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., at least one insertion site disclosed herein) in the FVIII protein. Non-limiting examples of the insertion sites are described elsewhere herein.

In other embodiments, a chimeric protein of the invention comprises three polypeptide chains, wherein a first polypeptide chain comprises, consists essentially of, or consists of a heavy chain of a FVIII protein, a second polypeptide chain comprises, consists essentially of, or consists of a light chain of a FVIII protein fused to a first Ig constant region or a portion thereof (e.g., a first Fc region), and a third polypeptide chain comprises, consists essentially of, or consists of a VWF fragment comprising a D' domain and a D3 domain, an XTEN sequence, a second Ig constant region or a portion thereof (e.g., a second Fc region), and an optional linker between the XTEN sequence and the second Ig constant region or a portion thereof or the VWF fragment and the XTEN sequence. The linker in the third chain can be a cleavable linker, which is cleaved at the site of coagulation, e.g., a thrombin cleavage site. In some embodiments, the heavy chain FVIII or the light chain FVIII is linked to one or more XTEN sequences, which can be linked to the N-terminus, the C-terminus, or inserted within one or more insertion sites within the FVIII sequence. Non-limiting examples of the insertion sites are disclosed elsewhere herein.

In yet other embodiments, a chimeric protein of the invention comprises two polypeptide chains, a first polypeptide chain comprising, consisting essentially of, or consisting of a heavy chain of a FVIII protein and a second polypeptide chain comprising, consisting essentially of, or consisting of a light chain of a FVIII protein, a first Ig constant region or a portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker, which contains one or more protease cleavage sites comprising one or more intracellular processing sites), a VWF fragment, a second linker (e.g., a thrombin cleavable linker), an XTEN sequence, and a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the light chain of the FVIII protein is linked to the first Ig constant region or a portion thereof (e.g., the first Fc region), which is further linked to the VWF fragment by the first linker, and wherein the VWF fragment is linked to the XTEN sequence, which is further linked to the second Ig constant

-492018203206 08 May 2018 [0118] [0119] region or a portion thereof by the second linker. In certain embodiments, the first linker is a processable linker, and the second linker is a cleavable linker. Upon expression, the chimeric protein can be processed by an intracellular processing enzyme, which cleaves the processable linker, and thus the chimeric protein can comprise, consists essentially of, or consists of three polypeptide chains. In addition, the VWF fragment can be cleaved off at the site of coagulation due to the cleavable linker.

In certain embodiments, a chimeric protein of the invention comprises one polypeptide chain, which comprises a single chain FVIII protein, a first Ig constant region or a portion thereof (e.g., a first Fc region), a first linker (e.g., a processable linker), a VWF fragment, an XTEN sequence, a second linker (e.g., a thrombin cleavable linker), and a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the single chain FVIII protein is linked to the first Ig constant region or a portion thereof, which is also linked to the VWF fragment by the first linker, and the VWF fragment is linked to the XTEN sequence, which is further linked to the second Ig constant region or a portion thereof. In one embodiment, the VWF fragment and the XTEN sequence are linked by the second linker. In another embodiment, the XTEN sequence and the second Ig constant region or a portion thereof are linked by the second linker. In other embodiments, the second chain further comprises a third linker. The single polypeptide chain can thus comprise the VWF fragment linked to the XTEN sequence by the second linker and the XTEN linked to the second Ig constant region or a portion thereof by the third linker. The second linker and the third linker can be identical or different. In one embodiment, the first linker is a processable linker. In another embodiment, the second linker or the third linker is a cleavable linker comprising one or two cleavable sites. In a specific embodiment, the second linker is a thrombin cleavable linker. The linkers useful in the invention are described elsewhere herein.

(2) FVIII, XTEN, and Fc

A chimeric protein of the invention also comprises (i) a FVIII protein, (ii) an XTEN sequence (a first half-life extender), and (iii) an Ig constant region or a portion thereof (a second half-life extender), in which the XTEN sequence is linked to the FVIII protein by an optional linker and the Ig constant region or a

-502018203206 08 May 2018 [0120] [0121] portion thereof by an additional optional linker. The XTEN sequence and the Ig constant region or a portion thereof can be used together to extend half-life of the FVIII protein. In one embodiment, the chimeric protein is a monomer. In another embodiment, the chimeric protein is a dimer (a homodimer or a heterodimer).

The present invention is also directed to a chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) an Ig constant region or a portion thereof (i.e., a first Ig constant region or a portion thereof, F, or FI), and (iv) an additional Ig constant region or a portion thereof (i.e., a second Ig constant region or a portion thereof or F2). In one embodiment, the XTEN sequence is linked to the FVIII protein at the C-terminus or the N-terminus or inserted immediately downstream of one or more amino acids in the FVIII protein (e.g., one or more XTEN insertion sites), the FVIII protein is linked to the first Ig constant region or a portion thereof, and the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are associated with or linked to each other by an optional linker. In certain aspects, the chimeric protein is a monomer-dimer hybrid, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a FVIII protein, an XTEN sequence, and a first Ig constant region or a portion thereof, and the second polypeptide chain comprises, consists essentially of, or consists of a second Ig constant region or a portion thereof without the FVIII protein and wherein the first chain and the second chain are associated with each other. The association between the Ig constant region or a portion thereof (e.g., the first Fc region) and the additional Ig constant region or a portion thereof (e.g., a second Fc region) is a chemical association or a physical association. In certain embodiments, the chemical association is a covalent bond. In other embodiments, the chemical association is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, the association is a non-peptide covalent bond. In still other embodiments, the association is a peptide bond.

In other aspects, the chimeric protein is a single polypeptide chain comprising a FVIII protein, an XTEN sequence, a first Ig constant region or a portion thereof, a linker, e.g., a processable linker, and a second Ig constant region

-51 2018203206 08 May 2018 [0122] [0123] [0124] [0125] or a portion thereof, wherein the single polypeptide chain is processed after expression by an intracellular enzyme and becomes two polypeptide chains.

In one embodiment, the lg constant region or a portion thereof (sometimes indicated herein by F or FI) linked to the FVIII protein can extend the half-life of the FVIII protein together with the XTEN sequence. In another embodiment, the lg constant region or a portion thereof (F or FI) is an Fc region or an FcRn binding partner described elsewhere herein.

In other embodiments, the additional lg constant region or a portion thereof (sometimes indicated herein by F2 or a second lg constant region or a portion thereof) associated with or linked to the first lg constant region or a portion thereof can also extend the half-life of the FVIII protein. In other embodiments, the second lg constant region or a portion thereof (F2) together with the first lg constant region or a portion thereof and the XTEN sequence can extend the half-life of the FVIII protein. The additional lg constant region or a portion thereof can be an Fc region or an FcRn binding partner described elsewhere herein.

In certain embodiments, the second lg constant region or a portion thereof associated with the first lg constant region or a portion thereof is further linked to a VWF fragment described elsewhere herein and an optional XTEN sequence.

In some embodiments, either or both of the lg constant region or a portion thereof (F or FI or a first lg constant region or a portion thereof) and an additional lg constant region or a portion thereof (i.e., a second lg constant region or a portion thereof or F2) (indicated in this paragraph as the lg constant regions or portion thereof') can include, but not limited to, a CHI domain, a CH2 domain, a CH3 domain, a CH4 domain, a hinge domain, any functional fragments, derivatives, or analogs thereof or two or more combinations thereof. In one embodiment, the lg constant region or a portion thereof comprises at least one CHI domain, at least one CH2 domain, at least one CH3 domain, at least one CH4 domain, or the functional fragments, derivatives, or analogues thereof. In another embodiment, the lg constant region or a portion thereof comprises at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in the hinge-CH2 orientation). In other embodiments, the lg constant domain or portion thereof comprises at least one CH2 domain or portion thereof

-522018203206 08 May 2018 [0126] [0127] and at least one CH3 domain or portion thereof (e.g., in the CF2-CH3 orientation).

Examples of the combination include, but are not limited to, a CH2 domain, a

CH3 domain, and a hinge domain, which are also known as an Fc region (or Fc domain), e.g., first Fc region. Additional examples of the lg constant regions or portion thereof are described elsewhere herein.

The chimeric protein of the invention can have an extended half-life of the FVIII protein compared to wild-type FVIII. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the half-life of wild type FVIII. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

(3) FVIII, XTEN, and VWF

In one aspect, a chimeric protein of the present invention comprises (i) a FVIII protein, (ii) an XTEN sequence, and (iii) a VWF fragment comprising a D' domain and a D3 domain of VWF, wherein the FVIII protein is linked to the XTEN sequence and wherein the FVIII protein is associated with or linked to the VWF fragment. In one embodiment, the VWF fragment of the chimeric protein described herein is not capable of binding to a VWF clearance receptor. In another embodiment, the VWF fragment is capable of protecting the FVIII protein from one or more protease cleavages, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors. In other embodiments, the VWF fragment prevents or inhibits binding of endogenous VWF to the VWF binding site in the FVIII protein. The VWF binding site can be located in the A3 domain or the C2 domain of the FVIII protein or both the A3

-53 2018203206 08 May 2018 [0128] [0129] domain and the C2 domain. In a specific embodiment, the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and/or amino acids 2303 to 2332 of SEQ ID NO: 2.

In another aspect, a chimeric protein comprises (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, which comprises a D' domain and a D3 domain of VWF, and (iv) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein at the C-terminus or the N-terminus or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein, the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the Ig constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof. The Ig constant region or a portion thereof useful for chimeric proteins of the invention is described elsewhere herein. In one embodiment, the Ig constant region or a portion thereof is capable of extending the half-life of a FVIII protein. In another embodiment, the Ig constant region or a portion thereof comprises a first Fc region or a first FcRn binding partner. In yet other embodiments, the Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker. In still other embodiments, the linker comprises a cleavable linker. The chimeric protein can be a single polypeptide chain, i.e., a monomer (/.e., a single chain), containing (i) , (ii), (iii), and (iv) or two chains containing a first chain comprising (i) and (ii) and a second chain comprising (iii) and (iv). In other aspects, the chimeric protein is a dimer (e.g., a homodimer or a heterodimer). In one embodiment, the chimeric protein comprises two chains, each comprising (i), (ii), (iii), and (iv).

In certain embodiments, a chimeric protein comprises (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, which comprises a D' domain and a D3 domain of VWF, (iv) an Ig constant region or a portion thereof (sometimes also indicated as F, a first Ig constant region or a portion thereof', or F2), and (v) an additional Ig constant region or a portion thereof (sometimes also indicated as F2 or a second Ig constant region or a portion thereof'), wherein (1) the FVIII protein is linked to the XTEN sequence at the C-terminus or N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein, (2)

-542018203206 08 May 2018 [0130] either the XTEN sequence or the FVIII protein is linked to the Ig constant region or a portion thereof, (3) the VWF fragment is linked to the second Ig constant region or a portion thereof, and (4) the Ig constant region or a portion thereof is associated with the second Ig constant region or a portion thereof. In one embodiment, the Ig constant region or a portion thereof linked to the FVII protein or the XTEN sequence is further linked to the VWF fragment by a linker, e.g., a processable linker. In another embodiment, the additional Ig constant region or a portion thereof useful for chimeric proteins of the invention can further be linked to the FVIII protein or the Ig constant region or a portion thereof by an optional linker, e.g., a processable linker. In some embodiments, a pair of the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof, each of which are linked to the VWF fragment and the FVIII protein, provides a bond stronger than the non-covalent bond between the FVIII protein and the VWF fragment, i.e., a covalent bond, e.g., a disulfide bond, thereby preventing endogenous VWF from replacing the VWF fragment in vivo. In other embodiments, either or both of the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are capable of extending a halflife of the FVIII protein or the VWF fragment. In other embodiments, the additional Ig constant region or a portion thereof comprises a second Fc region or an FcRn binding partner. The Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof in the chimeric proteins are identical or different.

In certain embodiments, the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are associated by a chemical association or a physical association. In one embodiment, the chemical association, i.e., (:), is at least one non-peptide bond. In certain embodiments, the chemical association, i.e., (:), is a covalent bond. In other embodiments, the chemical association, i.e., (:), is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a non-peptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence

-55 2018203206 08 May 2018 [0131] such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety. In some embodiments, the association between the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof can be a covalent bond, e.g., a disulfide bond, which prevents replacement the VWF fragment or the polypeptide containing the VWF fragment with endogenous VWF. Therefore, preventing interaction between the FVIII protein and endogenous VWF reduces or eliminates this half-life limiting factor for the FVIII protein, and thus the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF protein or wild-type FVIII.

In other aspects, a chimeric protein comprises a formula comprising:

(1) F VIII(X 1 )-L 1 -F1: V-L2-X2-L3 -F2;

(2) FVIII(X1)-L1-F1 :F2-L3-X2-L2-V;

(3) F1 -L1 -FVIII(X 1): V-L2-X2-L3 -F2;

(4) F1 -L1 -FVIII(X 1) :F2-L3 -X2-L2-V;

(5) FVIII(X1)-L1 -F1-L4-V-L2-X2-L3-F2;

(6) FVIII(X1 )-L 1 -F1-L4-F2-L3-X2-L2-V;

(7) F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (8) F1-L1-FVIII(X1)-L4- F2-L3-X2-L2-V, wherein FVIII(Xl) comprises a FVIII protein and one or more XTEN sequences, wherein the one or more XTEN sequence are linked to the N-terminus or Cterminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites disclosed herein) in the FVIII protein;

each of LI, L2, or L3 comprises an optional linker, e.g., a cleavable linker;

L4 is a linker, e.g., a processable linker;

X2 comprises one or more optional XTEN sequences;

FI comprises an Ig constant region or a portion thereof;

F2 comprises an optional additional Ig constant region or a portion thereof, and V comprises a VWF fragment;

-562018203206 08 May 2018 [0132] (-) is a peptide bond or one or more amino acids; and (:) comprises a chemical association or a physical association. In one embodiment, (:) represents a chemical association, e.g., at least one non-peptide bond. In another embodiment, the chemical association, i.e., (:) is a covalent bond. In other embodiments, the chemical association, i.e., (:) is a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In other embodiments, (:) is a nonpeptide covalent bond. In still other embodiments, (:) is a peptide bond. In yet other embodiments, (:) represents a physical association between two sequences, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety, and further that this physical association is maintained without allowing the second sequence to interact with other moieties. The orientation of the polypeptide formulas herein is listed from N-terminus (left) to C-terminus (right). For example, formula V-X-FVIII means formula NH2-VX-FVIII -COOH. In one embodiment, the formulas described herein can comprise any additional sequences between the two moieties. For example, formula V-XFVIII can further comprise any sequences at the N-terminus of V between V and X, between X and FVIII, or at the C-terminus of FVIII unless otherwise specified. In another embodiment, the hyphen (-) indicates a peptide bond.

In one aspect, the chimeric protein comprises two polypeptide chains, (A) a first chain comprising (i) a single chain FVIII protein (ii) an XTEN sequence, and (iii) a first Ig constant region or a portion thereof, e.g., a first Fc region or FcRn binding partner, wherein the XTEN sequence is linked to the FVIII protein at the N-terminus or C-terminus or inserted immediately downstream of one or more amino acids of the FVIII protein (e.g., one or more XTEN insertion sites disclosed herein) and the first Ig constant region or a portion thereof is linked to the XTEN sequence when the XTEN sequence is linked to the FVIII protein at the N-terminus or the C-terminus or the FVIII protein when the XTEN sequence is inserted within the FVIII protein, and (B) a second chain comprising (iv) a VWF fragment comprising a D' domain and a D3 domain, (v) a linker, and (vi) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein the VWF fragment is linked to the linker, e.g., a

-572018203206 08 May 2018 [0133] [0134] cleavable linker, which is further linked to the second Ig constant region or a portion thereof, and wherein the first polypeptide chain and the second polypeptide chain are associated with each other, e.g., a covalent bond, e.g., a disulfide bond. In one embodiment, the linker is a cleavable linker described elsewhere herein, e.g., a thrombin cleavable linker. In some embodiments, the second chain comprises one or more XTEN sequences between (iv) and (v) or (v) and (vi).

In other aspects, the chimeric protein comprises one polypeptide chain comprising (i) a single chain FVIII protein (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof, e.g., a first Fc region or a first FcRn binding partner, (iv) a first linker, (v) a VWF fragment comprising a D' domain and a D3 domain, (vi) a second linker, and (vii) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein (i) to (vii) are linked in the order or in any orders. In one embodiment, the first linker is a processable linker, which can be intracellularly processed or cleaved after expression and makes the single polypeptide chain into two polypeptide chains.

In another embodiment, the second linker is a cleavable linker described herein, e.g., a thrombin cleavable linker. The XTEN sequence used herein can be linked to the FVIII protein by an optional linker at the N-terminus or the C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein.

In certain aspects, a chimeric protein comprises three polypeptide chains, (A) a first polypeptide chain comprising (i) a heavy chain of a FVIII protein and (ii) an XTEN sequence, which are linked to each other and (B) a second polypeptide chain comprising (iii) a light chain of the FVIII protein and (iv) a first Ig constant region or a portion thereof, e.g., a first Fc region or a first FcRn binding partner, which are linked to each other, and (C) a third polypeptide chain comprising (v) a VWF fragment comprising a D' domain and a D3 domain, (vi) a linker, and (vii) a second Ig constant region or a portion thereof, e.g., a second Fc region or a second FcRn binding partner, wherein the second chain is associated with the first chain and the third chain. In one embodiment, the association between the first chain and the second chain is a chemical association or a physical association. For example, the association between the first chain and the

-582018203206 08 May 2018 [0135] [0136] second chain can be a metal bond. In another embodiment, the association between the second chain and the third chain is also a chemical association or a physical association, e.g., a covalent bond or a non-covalent bond. In certain embodiments, the association between the second chain and the third chain is through the two Ig constant regions or a portion thereof and is a disulfide bond. The bonding between the second chain and the third chain prevents or inhibits binding of the FVIII protein with endogenous VWF, thus preventing the FVIII protein being cleared by the VWF clearance pathway. In some embodiments, the linker is a processable linker, which is intracellularly cleaved after expression in a host cell. The XTEN sequence used herein is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein.

In certain embodiments, the VWF fragment is directly linked to the FVIII protein, which comprises one or more XTENs, by a peptide bond or a linker. As one way of linking the VWF fragment and the FVIII protein, in which one or more XTENs are inserted or linked, through a direct link (e.g. a peptide bond) or a linker, an enzymatic ligation (e.g., sortase) can be employed. For example, sortase refers to a group of prokaryotic enzymes that modify surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. For most substrates of sortase enzymes, the recognition signal consists of the motif LPXTG (Leu-Proany-Thr-Gly (SEQ ID NO: 51), then a highly hydrophobic transmembrane sequence, then a cluster of basic residues such as arginine. Cleavage occurs between the Thr and Gly, with transient attachment through the Thr residue to the active site Cys residue of a ligation partner, followed by transpeptidation that attaches the protein covalently to the cell wall. In some embodiments, the ligation partner contains Gly(n). In other embodiments, the chimeric protein further comprises a sortase recognition motif. In some embodiments, the VWF fragment is attached to FVIII comprising one or more XTENs inserted within or linked to using sortase mediated in vitro protein ligation.

In one embodiment, a VWF fragment linked to a sortase recognition motif by an optional linker can be fused to a FVIII protein linked to Gly(n) by a sortase, wherein n can be any integer and wherein one or more XTENs are inserted within

-592018203206 08 May 2018 [0137] or linked to the FVIII protein. A ligation construct comprises the VWF fragment (N-terminal portion of the construct) and the FVIII protein, in which one or more XTENs are inserted or linked (C-terminal portion of the construct), wherein the sortase recognition motif is inserted in between. Another ligation construct comprises the VWF fragment (N-terminal portion of the construct, the linker, the sortase recognition motif, and the FVIII protein, in which one or more XTENs are inserted or linked (C-terminal portion of the construct). In another embodiment, a FVIII protein linked to a sortase recognition motif by an optional linker can be fused to a VWF fragment linked to Gly(n) by a sortase, wherein n is any integer.

A resulting ligation construct comprises the FVIII protein (N-terminal portion of the construct), in which one or more XTENs are inserted or linked, and the VWF fragment (C-terminal portion of the construct), wherein the sortase recognition motif is inserted in between. Another resulting ligation construct comprises the FVIII protein (N-terminal portion of the construct), in which one or more XTENs are inserted or linked, the linker, the sortase recognition motif, and the VWF fragment (C-terminal portion of the construct). In other embodiments, a VWF fragment linked to a sortase recognition motif by a first optional linker can be fused to a heterologous moiety, e.g., an immunoglobulin constant region or a portion thereof, e.g., an Fc region, linked to a thrombin cleavage site by a second optional linker. A resulting construct can comprise the VWF fragment (Nterminal portion), the first linker, the sortase recognition motif, the protease cleavage site, the second optional linker, and the heterologous moiety.

In some embodiments, the VWF fragment is associated with the FVIII protein. The association between the VWF fragment and the FVIII protein can be a chemical association or a physical association. The chemical association can be a non-covalent interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic interaction, a Van der Waals interaction, or a hydrogen bond. In yet other embodiments, the association between the FVIII protein and the VWF fragment is a physical association between two sequences, e.g., due to an additional association between the sequence having the FVIII protein and the sequence having the VWF fragment, wherein a portion of a first sequence is in close proximity to a second sequence such that the first sequence shields or blocks a portion of the second sequence from interacting with another moiety.

-602018203206 08 May 2018 [0138] [0139]

As a result of preventing or inhibiting endogenous VWF interaction with the FVIII protein by the VWF fragment, the chimeric protein described herein have an extended half-life compared to wild-type FVIII or the corresponding chimeric protein without the VWF fragment. In one embodiment, the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the VWF fragment. In another embodiment, the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In a particular embodiment, the half-life of the FVIII protein is extended at least 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, or at least about 27 hours in HemA mice.

A) Von Willebrand Factor (VWF) Fragments

VWF (also known as F8VWF) is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelian connective tissue. The basic VWF monomer is a 2813 amino acid protein. Every monomer contains a number of specific domains with a specific function, the D'/D3 domain (which binds to Factor VIII), the Al domain (which binds to platelet GPIbreceptor, heparin, and/or possibly collagen), the A3 domain (which binds to collagen), the Cl domain (in which the RGD domain binds to platelet integrin allbp3 when this is activated), and the cysteine knot domain at the C-terminal

-61 2018203206 08 May 2018 end of the protein (which VWF shares with platelet-derived growth factor (PDGF), transforming growth factor-β (TGF3) and β-human chorionic gonadotropin (βΗΟΟ).

[0140] The term a VWF fragment as used herein includes, but is not limited to, functional VWF fragments comprising a D' domain and a D3 domain, which are capable of inhibiting binding of endogenous VWF to FVIII. In one embodiment, the VWF fragment binds to the FVIII protein. In another embodiment, the VWF fragment blocks the VWF binding site on the FVIII protein, thereby inhibiting interaction of the FVIII protein with endogenous VWF. The VWF fragments include derivatives, variants, mutants, or analogues that retain these activities of VWF.

[0141] The 2813 monomer amino acid sequence for human VWF is reported as

Accession Number NP 000543.2_in Genbank. The nucleotide sequence encoding the human VWF is reported as Accession Number_NM_000552.3_ in

Genbank. The nucleotide sequence of human VWF is designated as SEQ ID NO: 1. SEQ ID NO: 2 is the amino acid sequence encoded by SEQ ID NO: 1. Each domain of VWF is listed in Table 1.

TABLE 1. VWF Sequences

VWF domains	Amino acid Sequence
VWF Signal Peptide	1	MIPARFAGVL LALALILPGT LC
(Amino acids 1 to 22		22
of SEQ ID NO :2)
VWF D1D2 region	23			AEGTRGRS
		STARCSLFGS	DFVNTFDGSM
(Amino acids 23 to	51	YSFAGYCSYL	LAGGCQKRSF	SIIGDFQNGK
763 of SEQ ID NO: 2)		RVSLSVYLGE	FFDIHLFVNG
	101	TVTQGDQRVS	MPYASKGLYL	ETEAGYYKLS
		GEAYGFVARI	DGSGNFQVLL
	151	SDRYFNKTCG	LCGNFNIFAE	DDFMTQEGTL
		TSDPYDFANS	WALSSGEQWC
	201	ERASPPSSSC	NISSGEMQKG	LWEQCQLLKS
		TSVFARCHPL	VDPEPFVALC
	251	EKTLCECAGG	LECACPALLE	YARTCAQEGM
		VLYGWTDHSA	CSPVCPAGME
	301	YRQCVSPCAR	TCQSLHINEM	CQERCVDGCS
		CPEGQLLDEG	LCVESTECPC
	351	VHSGKRYPPG	TSLSRDCNTC	ICRNSQWICS
		NEECPGECLV	TGQSHFKSFD
	401	NRYFTFSGIC	QYLLARDCQD	HSFSIVIETV

-622018203206 08 May 2018

QCADDRDAVC TRSVTVRLPG 4 51 LHNSLVKLKH GAGVAMDGQD

RIQHTVTASV RLSYGEDLQM 501 DWDGRGRLLV KLSPVYAGKT

QGDDFLTPSG LAEPRVEDFG 551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS

EEACAVLTSP TFEACHRAVS 601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA

AACAGRGVRV AWREPGRCEL

IQLPLLKGDL

CGLCGNYNGN

	6 51 701 751 7 63	NCPKGQVYLQ EACLEGCFCP CVPKAQCPCY CEDGFMHCTM AVLSSPLSHR	CGTPCNLTCR PGLYMDERGD YDGEIFQPED SGVPGSLLPD	SLSYPDEECN IFSDHHTMCY SKR
VWF D' Domain	7 64		SLSCRPP MVKLVCPADN
	LRAEGLECTK
		TCONYDLECM
	SOI	SMGCVSGCLC	PPGMVRHENR	CVALERCPCF
		HQGKEYAPGE	TVKIGCNTCV
	S 51	CRDRKWNCTD		HVCDAT
		8 6 6
VWF D3 Domain	867		CSTI GMAHYLTFDG
		LKYLFPGECQ	YVLVQDYCGS
	901	NPGTFRILVG	NKGCSHPSVK	CKKRVTILVE
		GGEIELFDGE	VNVKRPMKDE
	951	THFEVVESGR	YIILLLGKAL	SVVWDRHLSI
		SVVLKQTYQE	KVCGLCGNFD
	10 01	GIQNNDLTSS	NLQVEEDPVD	FGNSWKVSSQ
		CADTRKVPLD	SSPATCHNNI
	10 51	MKQTMVDSSC	RILTSDVFQD	CNKLVDPEPY
		LDVCIYDTCS	CESIGDCACF
	1101	CDTIAAYAHV	CAQHGKVVTW	RTATLCPQSC
		EERNLRENGY	ECEWRYNSCA
	1151	PACQVTCQHP	EPLACPVQCV	EGCHAHCPPG
		KILDELLQTC	VDPEDCPVCE
	12 01	VAGRRFASGK	KVTLNPSDPE HCQICHCDVV
	1240	NLTCEACQEP
VWF Al Domain	1241	GGLVVPPTDA
	1251	PVSPTTLYVE	DISEPPLHDF	YCSRLLDLVF
		LLDGSSRLSE	AEFEVLKAFV
	13 01	VDMMERLRIS	QKWVRVAVVE	YHDGSHAYIG
		LKDRKRPSEL	RRIASQVKYA
	13 51	GSQVASTSEV	LKYTLFQIFS	KIDRPEASRI
		ALLLMASQEP	QRMSRNFVRY
	1401	VQGLKKKKVI	VIPVGIGPHA	NLKQIRLIEK

-63 2018203206 08 May 2018

	QAPENKAFVL	SSVDELEQQR
14 51	DEIVSYLCDL	APEAPPPTLP	PDMAQVTVG
	1479
14 8 0			P
	GLLGVSTLGP	KRNSMVLDVA
1501	FVLEGSDKIG	EADFNRSKEF	MEEVIQRMDV
	GQDSIHVTVL	QYSYMVTVEY
1551	PFSEAQSKGD	ILQRVREIRY	QGGNRTNTGL
	ALRYLSDHSF	LVSQGDREQA
	1600
1601	PNLVYMVTGN	PASDEIKRLP	GDIQVVPIGV
	GPNANVQELE	RIGWPNAPIL
1651	IQDFETLPRE	APDLVLQRCC	SGEGLQIPTL
	SPAPDCSQPL	DVILLLDGSS
17 01	SFPASYFDEM	KSFAKAFISK	ANIGPRLTQV
	SVLQYGSITT	IDVPWNVVPE
1Ί 51	KAHLLSLVDV	MQREGGPSQI	GDALGFAVRY
	LTSEMHGARP	GASKAVVILV
18 01	TDVSVDSVDA	AADAARSNRV	TVFPIGIGDR
	YDAAQLRILA	GPAGDSNVVK
1851	LQRIEDLPTM	VTLGNSFLHK	LCSGFVRICM
	DEDGNEKRPG	DVWTLPDQCH
1901	TVTCQPDGQT	LLKSHRVNCD	RGLRPSCPNS
	QSPVKVEETC	GCRWTCPCVC
1951	TGSSTRHIVT	FDGQNFKLTG	SCSYVLFQNK
	EQDLEVILHN	GACSPGARQG
2001	CMKSIEVKHS	ALSVEXHSDM	EVTVNGRLVS
	VPYVGGNMEV	NVYGAIMHEV
2 0 51	RFNHLGHIFT	FTPQNNEFQL	QLSPKTFASK
	TYGLCGICDE	NGANDFMLRD
2101	GTVTTDWKTL	VQEWTVQRPG	QTCQPILEEQ
	CLVPDSSHCQ	VLLLPLFAEC
2151	HKVLAPATFY	AICQQDSCHQ	EQVCEVIASY
	AHLCRTNGVC	VDWRTPDFCA
22 01	MSCPPSLVYN	HCEHGCPRHC	DGNVSSCGDH
	PSEGCFCPPD	KVMLEGSCVP
2251	EEACTQCIGE	DGVQHQFLEA	WVPDHQPCQI
	CTCLSGRKVN	CTTQPCPTAK
2301	APTCGLCEVA	RLRQNADQCC	PEYECVCDPV
	SCDLPPVPHC	ERGLQPTLTN
2 3 51	PGECRPNFTC	ACRKEECKRV	SPPSCPPHRL
	PTLRKTQCCD	EYECACNCVN
2401	STVSCPLGYL	ASTATNDCGC	TTTTCLPDKV
	CVHRSTIYPV	GQFWEEGCDV
2451	CTCTDMEDAV	MGLRVAQCSQ	KPCEDSCRSG
	FTYVLHEGEC	CGRCLPSACE
2501	VVTGSPRGDS	QSSWKSVGSQ	WASPENPCLI
	NECVRVKEEV	FIQQRNVSCP
2551	QLEVPVCPSG	FQLSCKTSAC	CPSCRCERME
	ACMLNGTVIG	PGKTVMIDVC

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	2601 TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC CGRCLPTACT 2651 IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2701 CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC 2751 QGKCASKAMY SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSVVYHEVLN 2801 AMECKCSPRK CSK
	Nucleotide Sequence (SEQ ID NO: 1)
Full-length VWF	1 ATGATTCCTG CCAGATTTGC CGGGGTGCTG CTTGCTCTGG CCCTCATTTT 51 GCCAGGGACC CTTTGTGCAG AAGGAACTCG CGGCAGGTCA TCCACGGCCC 101 GATGCAGCCT TTTCGGAAGT GACTTCGTCA ACACCTTTGA TGGGAGCATG 151 TACAGCTTTG CGGGATACTG CAGTTACCTC CTGGCAGGGG GCTGCCAGAA 201 ACGCTCCTTC TCGATTATTG GGGACTTCCA GAATGGCAAG AGAGTGAGCC 251 TCTCCGTGTA TCTTGGGGAA TTTTTTGACA TCCATTTGTT TGTCAATGGT 301 ACCGTGACAC AGGGGGACCA AAGAGTCTCC ATGCCCTATG CCTCCAAAGG 351 GCTGTATCTA GAAACTGAGG CTGGGTACTA CAAGCTGTCC GGTGAGGCCT 401 ATGGCTTTGT GGCCAGGATC GATGGCAGCG GCAACTTTCA AGTCCTGCTG 4 51 T CAGACAGAT AC T T CAACAA GACCTGCGGG CTGTGTGGCA ACTTTAACAT 501 CTTTGCTGAA GATGACTTTA TGACCCAAGA AGGGACCTTG ACCTCGGACC 551 CTTATGACTT TGCCAACTCA TGGGCTCTGA GCAGTGGAGA ACAGTGGTGT 601 GAACGGGCAT CTCCTCCCAG CAGCTCATGC AACATCTCCT CTGGGGAAAT 651 GCAGAAGGGC CTGTGGGAGC AGTGCCAGCT TCTGAAGAGC ACCTCGGTGT 701 TTGCCCGCTG CCACCCTCTG GTGGACCCCG AGCCTTTTGT GGCCCTGTGT 751 GAGAAGACTT TGTGTGAGTG TGCTGGGGGG CTGGAGTGCG CCTGCCCTGC 801 CCTCCTGGAG TACGCCCGGA CCTGTGCCCA GGAGGGAATG GTGCTGTACG 851 GCTGGACCGA CCACAGCGCG TGCAGCCCAG TGTGCCCTGC TGGTATGGAG 901 TATAGGCAGT GTGTGTCCCC TTGCGCCAGG ACCTGCCAGA GCCTGCACAT 951 CAATGAAATG TGTCAGGAGC

-65 2018203206 08 May 2018

GATGCGTGGA TGGCTGCAGC TGCCCTGAGG 1001 GACAGCTCCT GGATGAAGGC CTCTGCGTGG AGAGCACCGA GTGTCCCTGC 1051 GTGCATTCCG GAAAGCGCTA CCCTCCCGGC ACCTCCCTCT CTCGAGACTG 1101 CAACACCTGC ATTTGCCGAA ACAGCCAGTG GATCTGCAGC AATGAAGAAT 1151 GTCCAGGGGA GTGCCTTGTC ACTGGTCAAT CCCACTTCAA GAGCTTTGAC 1201 AACAGATACT TCACCTTCAG TGGGATCTGC CAGTACCTGC TGGCCCGGGA 1251 TTGCCAGGAC CACTCCTTCT CCATTGTCAT TGAGACTGTC CAGTGTGCTG 1301 ATGACCGCGA CGCTGTGTGC ACCCGCTCCG TCACCGTCCG GCTGCCTGGC 1351 CTGCACAACA GCCTTGTGAA ACTGAAGCAT GGGGCAGGAG TTGCCATGGA 1401 TGGCCAGGAC ATCCAGCTCC CCCTCCTGAA AGGTGACCTC CGCATCCAGC 1451 ATACAGTGAC GGCCTCCGTG CGCCTCAGCT ACGGGGAGGA CCTGCAGATG 1501 GACTGGGATG GCCGCGGGAG GCTGCTGGTG AAGCTGTCCC CCGTCTATGC 1551 CGGGAAGACC TGCGGCCTGT GTGGGAATTA CAATGGCAAC CAGGGCGACG 1601 ACTTCCTTAC CCCCTCTGGG CTGGCRGAGC CCCGGGTGGA GGACTTCGGG 1651 AACGCCTGGA AGCTGCACGG GGACTGCCAG GACCTGCAGA AGCAGCACAG 1701 CGATCCCTGC GCCCTCAACC CGCGCATGAC CAGGTTCTCC GAGGAGGCGT 1751 GCGCGGTCCT GACGTCCCCC ACATTCGAGG CCTGCCATCG TGCCGTCAGC 1801 CCGCTGCCCT ACCTGCGGAA CTGCCGCTAC GACGTGTGCT CCTGCTCGGA 1851 CGGCCGCGAG TGCCTGTGCG GCGCCCTGGC CAGCTATGCC GCGGCCTGCG 1901 CGGGGAGAGG CGTGCGCGTC GCGTGGCGCG AGCCAGGCCG CTGTGAGCTG 1951 AACTGCCCGA AAGGCCAGGT GTACCTGCAG TGCGGGACCC CCTGCAACCT 2001 GACCTGCCGC TCTCTCTCTT ACCCGGATGA GGAATGCAAT GAGGCCTGCC 2051 TGGAGGGCTG CTTCTGCCCC CCAGGGCTCT ACATGGATGA GAGGGGGGAC 2101 TGCGTGCCCA AGGCCCAGTG CCCCTGTTAC TATGACGGTG AGATCTTCCA 2151 GCCAGAAGAC ATCTTCTCAG ACCATCACAC CATGTGCTAC TGTGAGGATG 2201 GCTTCATGCA CTGTACCATG AGTGGAGTCC CCGGAAGCTT GCTGCCTGAC

-662018203206 08 May 2018

2251 GCTGTCCTCA GCAGTCCCCT GTCTCATCGC AGCAAAAGGA GCCTATCCTG 2301 TCGGCCCCCC ATGGTCAAGC TGGTGTGTCC CGCTGACAAC CTGCGGGCTG 2351 AAGGGCTCGA GTGTACCAAA ACGTGCCAGA ACTATGACCT GGAGTGCATG 2401 AGCATGGGCT GTGTCTCTGG CTGCCTCTGC CCCCCGGGCA TGGTCCGGCA 2451 TGAGAACAGA TGTGTGGCCC TGGAAAGGTG TCCCTGCTTC CATCAGGGCA 2501 AGGAGTATGC CCCTGGAGAA ACAGTGAAGA TTGGCTGCAA CACTTGTGTC 2551 TGTCGGGACC GGAAGTGGAA CTGCACAGAC CATGTGTGTG ATGCCACGTG 2601 CTCCACGATC GGCATGGCCC ACTACCTCAC CTTCGACGGG CTCAAATACC 2651 TGTTCCCCGG GGAGTGCCAG TACGTTCTGG TGCAGGATTA CTGCGGCAGT 2701 AACCCTGGGA CCTTTCGGAT CCTAGTGGGG AATAAGGGAT GCAGCCACCC 2751 CTCAGTGAAA TGCAAGAAAC GGGTCACCAT CCTGGTGGAG GGAGGAGAGA 2801 TTGAGCTGTT TGACGGGGAG GTGAATGTGA AGAGGCCCAT GAAGGATGAG 2851 ACTCACTTTG AGGTGGTGGA GTCTGGCCGG TACATCATTC TGCTGCTGGG 2901 CAAAGCCCTC TCCGTGGTCT GGGACCGCCA CCTGAGCATC TCCGTGGTCC 2951 TGAAGCAGAC ATACCAGGAG AAAGTGTGTG GCCTGTGTGG GAATTTTGAT 3001 GGCATCCAGA ACAATGACCT CACCAGCAGC AACCTCCAAG TGGAGGAAGA 3051 CCCTGTGGAC TTTGGGAACT CCTGGAAAGT GAGCTCGCAG TGTGCTGACA 3101 CCAGAAAAGT GCCTCTGGAC TCATCCCCTG CCACCTGCCA TAACAACATC 3151 ATGAAGCAGA CGATGGTGGA TTCCTCCTGT AGAATCCTTA CCAGTGACGT 3201 CTTCCAGGAC TGCAACAAGC TGGTGGACCC CGAGCCATAT CTGGATGTCT 3251 GCATTTACGA CACCTGCTCC TGTGAGTCCA TTGGGGACTG CGCCTGCTTC 3301 TGCGACACCA TTGCTGCCTA TGCCCACGTG TGTGCCCAGC ATGGCAAGGT 3351 GGTGACCTGG AGGACGGCCA CATTGTGCCC CCAGAGCTGC GAGGAGAGGA 3401 ATCTCCGGGA GAACGGGTAT GAGTGTGAGT GGCGCTATAA CAGCTGTGCA 3451 CCTGCCTGTC AAGTCACGTG TCAGCACCCT GAGCCACTGG CCTGCCCTGT 3501 GCAGTGTGTG GAGGGCTGCC

-672018203206 08 May 2018

ATGCCCACTG CCCTCCAGGG AAAATCCTGG 3551 ATGAGCTTTT GCAGACCTGC GTTGACCCTG AAGACTGTCC AGTGTGTGAG 3601 GTGGCTGGCC GGCGTTTTGC CTCAGGAAAG AAAGTCACCT TGAATCCCAG 3651 TGACCCTGAG CACTGCCAGA TTTGCCACTG TGATGTTGTC AACCTCACCT 3701 GTGAAGCCTG CCAGGAGCCG GGAGGCCTGG TGGTGCCTCC CACAGATGCC 3751 CCGGTGAGCC CCACCACTCT GTATGTGGAG GACATCTCGG AACCGCCGTT 3801 GCACGATTTC TACTGCAGCA GGCTACTGGA CCTGGTCTTC CTGCTGGATG 3851 GCTCCTCCAG GCTGTCCGAG GCTGAGTTTG AAGTGCTGAA GGCCTTTGTG 3901 GTGGACATGA TGGAGCGGCT GCGCATCTCC CAGAAGTGGG TCCGCGTGGC 3951 CGTGGTGGAG TACCACGACG GCTCCCACGC CTACATCGGG CTCAAGGACC 4001 GGAAGCGACC GTCAGAGCTG CGGCGCATTG CCAGCCAGGT GAAGTATGCG 4051 GGCAGCCAGG TGGCCTCCAC CAGCGAGGTC TTGAAATACA CACTGTTCCA 4101 AATCTTCAGC AAGATCGACC GCCCTGAAGC CTCCCGCATC GCCCTGCTCC 4151 TGATGGCCAG CCAGGAGCCC CAACGGATGT CCCGGAACTT TGTCCGCTAC 4201 GTCCAGGGCC TGAAGAAGAA GAAGGTCATT GTGATCCCGG TGGGCATTGG 4251 GCCCCATGCC AACCTCAAGC AGATCCGCCT CATCGAGAAG CAGGCCCCTG 4301 AGAACAAGGC CTTCGTGCTG AGCAGTGTGG ATGAGCTGGA GCAGCAAAGG 4351 GACGAGATCG TTAGCTACCT CTGTGACCTT GCCCCTGAAG CCCCTCCTCC 4401 TACTCTGCCC CCCGACATGG CACAAGTCAC TGTGGGCCCG GGGCTCTTGG 4451 GGGTTTCGAC CCTGGGGCCC AAGAGGAACT CCATGGTTCT GGATGTGGCG 4501 TTCGTCCTGG AAGGATCGGA CAAAATTGGT GAAGCCGACT TCAACAGGAG 4551 CAAGGAGTTC ATGGAGGAGG TGATTCAGCG GATGGATGTG GGCCAGGACA 4601 GCATCCACGT CACGGTGCTG CAGTACTCCT ACATGGTGAC CGTGGAGTAC 4651 CCCTTCAGCG AGGCACAGTC CAAAGGGGAC ATCCTGCAGC GGGTGCGAGA 4701 GATCCGCTAC CAGGGCGGCA ACAGGACCAA CACTGGGCTG GCCCTGCGGT 4751 ACCTCTCTGA CCACAGCTTC TTGGTCAGCC AGGGTGACCG GGAGCAGGCG

-682018203206 08 May 2018

4801 CCCAACCTGG TCTACATGGT CACCGGAAAT CCTGCCTCTG ATGAGATCAA 4851 GAGGCTGCCT GGAGACATCC AGGTGGTGCC CATTGGAGTG GGCCCTAATG 4901 CCAACGTGCA GGAGCTGGAG AGGATTGGCT GGCCCAATGC CCCTATCCTC 4951 ATCCAGGACT TTGAGACGCT CCCCCGAGAG GCTCCTGACC TGGTGCTGCA 5001 GAGGTGCTGC TCCGGAGAGG GGCTGCAGAT CCCCACCCTC TCCCCTGCAC 5051 CTGACTGCAG CCAGCCCCTG GACGTGATCC TTCTCCTGGA TGGCTCCTCC 5101 AGTTTCCCAG CTTCTTATTT TGATGAAATG AAGAGTTTCG CCAAGGCTTT 5151 CATTTCAAAA GCCAATATAG GGCCTCGTCT CACTCAGGTG TCAGTGCTGC 5201 AGTATGGAAG CATCACCACC ATTGACGTGC CATGGAACGT GGTCCCGGAG 5251 AAAGCCCATT TGCTGAGCCT TGTGGACGTC ATGCAGCGGG AGGGAGGCCC 5301 CAGCCAAATC GGGGATGCCT TGGGCTTTGC TGTGCGATAC TTGACTTCAG 5351 AAATGCATGG TGCCAGGCCG GGAGCCTCAA AGGCGGTGGT CATCCTGGTC 5401 ACGGACGTCT CTGTGGATTC AGTGGATGCA GCAGCTGATG CCGCCAGGTC 5451 CAACAGAGTG ACAGTGTTCC CTATTGGAAT TGGAGATCGC TACGATGCAG 5501 CCCAGCTACG GATCTTGGCA GGCCCAGCAG GCGACTCCAA CGTGGTGAAG 5551 CTCCAGCGAA TCGAAGACCT CCCTACCATG GTCACCTTGG GCAATTCCTT 5601 CCTCCACAAA CTGTGCTCTG GATTTGTTAG GATTTGCATG GATGAGGATG 5651 GGAATGAGAA GAGGCCCGGG GACGTCTGGA CCTTGCCAGA CCAGTGCCAC 5701 ACCGTGACTT GCCAGCCAGA TGGCCAGACC TTGCTGAAGA GTCATCGGGT 5751 CAACTGTGAC CGGGGGCTGA GGCCTTCGTG CCCTAACAGC CAGTCCCCTG 5801 TTAAAGTGGA AGAGACCTGT GGCTGCCGCT GGACCTGCCC CTGYGTGTGC 5851 ACAGGCAGCT CCACTCGGCA CATCGTGACC TTTGATGGGC AGAATTTCAA 5901 GCTGACTGGC AGCTGTTCTT ATGTCCTATT TCAAAACAAG GAGCAGGACC 5951 TGGAGGTGAT TCTCCATAAT GGTGCCTGCA GCCCTGGAGC AAGGCAGGGC 6001 TGCATGAAAT CCATCGAGGT GAAGCACAGT GCCCTCTCCG TCGAGSTGCA 6051 CAGTGACATG GAGGTGACGG

-692018203206 08 May 2018

TGAATGGGAG ACTGGTCTCT GTTCCTTACG 6101 TGGGTGGGAA CATGGAAGTC AACGTTTATG GTGCCATCAT GCATGAGGTC 6151 AGATTCAATC ACCTTGGTCA CATCTTCACA TTCACTCCAC AAAACAATGA 6201 GTTCCAACTG CAGCTCAGCC CCAAGACTTT TGCTTCAAAG ACGTATGGTC 6251 TGTGTGGGAT CTGTGATGAG AACGGAGCCA ATGACTTCAT GCTGAGGGAT 6301 GGCACAGTCA CCACAGACTG GAAAACACTT GTTCAGGAAT GGACTGTGCA 6351 GCGGCCAGGG CAGACGTGCC AGCCCATCCT GGAGGAGCAG TGTCTTGTCC 6401 CCGACAGCTC CCACTGCCAG GTCCTCCTCT TACCACTGTT TGCTGAATGC 6451 CACAAGGTCC TGGCTCCAGC CACATTCTAT GCCATCTGCC AGCAGGACAG 6501 TTGCCACCAG GAGCAAGTGT GTGAGGTGAT CGCCTCTTAT GCCCACCTCT 6551 GTCGGACCAA CGGGGTCTGC GTTGACTGGA GGACACCTGA TTTCTGTGCT 6601 ATGTCATGCC CACCATCTCT GGTCTACAAC CACTGTGAGC ATGGCTGTCC 6651 CCGGCACTGT GATGGCAACG TGAGCTCCTG TGGGGACCAT CCCTCCGAAG 6701 GCTGTTTCTG CCCTCCAGAT AAAGTCATGT TGGAAGGCAG CTGTGTCCCT 6751 GAAGAGGCCT GCACTCAGTG CATTGGTGAG GATGGAGTCC AGCACCAGTT 6801 CCTGGAAGCC TGGGTCCCGG ACCACCAGCC CTGTCAGATC TGCACATGCC 6851 TCAGCGGGCG GAAGGTCAAC TGCACAACGC AGCCCTGCCC CACGGCCAAA 6901 GCTCCCACGT GTGGCCTGTG TGAAGTAGCC CGCCTCCGCC AGAATGCAGA 6951 CCAGTGCTGC CCCGAGTATG AGTGTGTGTG TGACCCAGTG AGCTGTGACC 7001 TGCCCCCAGT GCCTCACTGT GAACGTGGCC TCCAGCCCAC ACTGACCAAC 7051 CCTGGCGAGT GCAGACCCAA CTTCACCTGC GCCTGCAGGA AGGAGGAGTG 7101 CAAAAGAGTG TCCCCACCCT CCTGCCCCCC GCACCGTTTG CCCACCCTTC 7151 GGAAGACCCA GTGCTGTGAT GAGTATGAGT GTGCCTGCAA CTGTGTCAAC 7201 TCCACAGTGA GCTGTCCCCT TGGGTACTTG GCCTCAACCG CCACCAATGA 7251 CTGTGGCTGT ACCACAACCA CCTGCCTTCC CGACAAGGTG TGTGTCCACC 7301 GAAGCACCAT CTACCCTGTG GGCCAGTTCT GGGAGGAGGG CTGCGATGTG

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7351 TGCACCTGCA CCGACATGGA

GGATGCCGTG ATGGGCCTCC GCGTGGCCCA

7401 GTGCTCCCAG AAGCCCTGTG

AGGACAGCTG TCGGTCGGGC TTCACTTACG

7451 TTCTGCATGA AGGCGAGTGC

TGTGGAAGGT GCCTGCCATC TGCCTGTGAG

7501 GTGGTGACTG GCTCACCGCG

GGGGGACTCC CAGTCTTCCT GGAAGAGTGT

7551 CGGCTCCCAG TGGGCCTCCC

CGGAGAACCC CTGCCTCATC AATGAGTGTG

7601 TCCGAGTGAA GGAGGAGGTC

TTTATACAAC AAAGGAACGT CTCCTGCCCC

7651 CAGCTGGAGG TCCCTGTCTG

CCCCTCGGGC TTTCAGCTGA GCTGTAAGAC

7701 CTCAGCGTGC TGCCCAAGCT

GTCGCTGTGA GCGCATGGAG GCCTGCATGC

7751 TCAATGGCAC TGTCATTGGG

CCCGGGAAGA CTGTGATGAT CGATGTGTGC

7801 ACGACCTGCC GCTGCATGGT

GCAGGTGGGG GTCATCTCTG GATTCAAGCT

7851 GGAGTGCAGG AAGACCACCT

GCAACCCCTG CCCCCTGGGT TACAAGGAAG

7901 AAAATAACAC AGGTGAATGT

TGTGGGAGAT GTTTGCCTAC GGCTTGCACC

7951 ATTCAGCTAA GAGGAGGACA

GATCATGACA CTGAAGCGTG ATGAGACGCT

8001 CCAGGATGGC TGTGATACTC

ACTTCTGCAA GGTCAATGAG AGAGGAGAGT

8051 ACTTCTGGGA GAAGAGGGTC

ACAGGCTGCC CACCCTTTGA TGAACACAAG

8101 TGTCTTGCTG AGGGAGGTAA

AATTATGAAA ATTCCAGGCA CCTGCTGTGA

8151 CACATGTGAG GAGCCTGAGT

GCAACGACAT CACTGCCAGG CTGCAGTATG

8201 TCAAGGTGGG AAGCTGTAAG

TCTGAAGTAG AGGTGGATAT CCACTACTGC

8251 CAGGGCAAAT GTGCCAGCAA

AGCCATGTAC TCCATTGACA TCAACGATGT

8301 GCAGGACCAG TGCTCCTGCT

GCTCTCCGAC ACGGACGGAG CCCATGCAGG

8351 TGGCCCTGCA CTGCACCAAT

GGCTCTGTTG TGTACCATGA GGTTCTCAAT

8401 GCCATGGAGT GCAAATGCTC CCCCAGGAAG TGCAGCAAGT GA [0142]

The VWF fragment as used herein can be a VWF fragment comprising a D' domain and a D3 domain of VWF, wherein the VWF fragment binds to Factor

-71 2018203206 08 May 2018 [0143]

VIII (FVIII) and inhibits binding of endogenous VWF (full-length VWF) to FVIII. The VWF fragment comprising the D' domain and the D3 domain can further comprise a VWF domain selected from the group consisting of an A1 domain, an A2 domain, an A3 domain, a DI domain, a D2 domain, a D4 domain, a BI domain, a B2 domain, a B3 domain, a Cl domain, a C2 domain, a CK domain, one or more fragments thereof, and any combinations thereof. In one embodiment, a VWF fragment comprises, consists essentially of, or consists of:

(1) the D' and D3 domains of VWF or fragments thereof; (2) the DI, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the DI, D2, D', and D3 domains of VWF or fragments thereof; or (5) the DI, D2, D', D3, and Al domains of VWF or fragments thereof. The VWF fragment described herein does not contain a site binding to a VWF clearance receptor. In another embodiment, the VWF fragment described herein is not amino acids 764 to 1274 of SEQ ID NO: 2. The VWF fragment of the present invention can comprise any other sequences linked to or fused to the VWF fragment. For example, a VWF fragment described herein can further comprise a signal peptide.

In one embodiment, the VWF fragment binds to or is associated with a FVIII protein. By binding to or associating with a FVIII protein, a VWF fragment of the invention protects FVIII from protease cleavage and FVIII activation, stabilizes the heavy chain and light chain of FVIII, and prevents clearance of FVIII by scavenger receptors. In another embodiment, the VWF fragment binds to or associates with a FVIII protein and blocks or prevents binding of the FVIII protein to phospholipid and activated Protein C. By preventing or inhibiting binding of the FVIII protein with endogenous, full-length VWF, the VWF fragment of the invention reduces the clearance of FVIII by VWF clearance receptors and thus extends half-life of the FVIII protein. In one embodiment, the half-life extension of a FVIII protein is thus due to the binding of or associating with the VWF fragment lacking a VWF clearance receptor binding site to the FVIII protein and shielding or protecting of the FVIII protein by the VWF fragment from endogenous VWF which contains the VWF clearance receptor binding site. The FVIII protein bound to or protected by the VWF fragment can also allow recycling of a FVIII protein. By eliminating the VWF clearance

-722018203206 08 May 2018 [0144] [0145] pathway receptor binding sites contained in the full length VWF molecule, the

FVIII/VWF heterodimers of the invention are shielded from the VWF clearance pathway, further extending FVIII half-life.

In one embodiment, a VWF fragment of the present invention comprises the D' domain and the D3 domain of VWF, wherein the D' domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In another embodiment, a VWF fragment comprises the D' domain and the D3 domain of VWF, wherein the D3 domain is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In some embodiments, a VWF fragment described herein comprises, consists essentially of, or consists of the D' domain and D3 domain of VWF, which are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In other embodiments, a VWF fragment comprises, consists essentially of, or consists of the DI, D2, D', and D3 domains at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 23 to 1240 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In still other embodiments, the VWF fragment further comprises a signal peptide operably linked thereto.

In some embodiments, a VWF fragment of the invention consists essentially of or consists of (1) the D’D3 domain, the D1DD3 domain, D2D’D3 domain, or D1D2DD3 domain and (2) an additional VWF sequence up to about 10 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1250 of SEQ ID NO: 2), up to about 15 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1255 of SEQ ID NO: 2), up to about 20 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2), up to about 25 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ ID NO: 2 to amino acids 764 to 1265 of SEQ ID NO: 2), or up to about 30 amino acids (e.g., any sequences from amino acids 764 to 1240 of SEQ

-73 2018203206 08 May 2018 [0146] [0147] [0148] [0149]

ID NO: 2 to amino acids 764 to 1260 of SEQ ID NO: 2). In a particular embodiment, the VWF fragment comprising or consisting essentially of the D' domain and the D3 domain is neither amino acids 764 to 1274 of SEQ ID NO: 2 nor the full-length mature VWF. In some embodiments, the D1D2 domain is expressed in trans with the D'D3 domain. In some embodiments, the D1D2 domain is expressed in cis with the D'D3 domain.

In other embodiments, the VWF fragment comprising the D'D3 domains linked to the D1D2 domains further comprises an intracellular cleavage site, e.g., (a cleavage site by PACE (furin) or PC5), allowing cleavage of the D1D2 domains from the D'D3 domains upon expression. Non-limiting examples of the intracellular cleavage site are disclosed elsewhere herein.

In yet other embodiments, a VWF fragment comprises the D' domain and the D3 domain, but does not comprise an amino acid sequence selected from the group consisting of (1) amino acids 1241 to 2813 of SEQ ID NO: 2, (2) amino acids 1270 to amino acids 2813 of SEQ ID NO: 2, (3) amino acids 1271 to amino acids 2813 of SEQ ID NO: 2, (4) amino acids 1272 to amino acids 2813 of SEQ ID NO: 2, (5) amino acids 1273 to amino acids 2813 of SEQ ID NO: 2, (6) amino acids 1274 to amino acids 2813 of SEQ ID NO: 2, and any combinations thereof

In still other embodiments, a VWF fragment of the present invention comprises, consists essentially of, or consists of an amino acid sequence corresponding to the D' domain, D3 domain, and Al domain, wherein the amino acid sequence is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid 764 to 1479 of SEQ ID NO: 2, wherein the VWF fragment prevents binding of endogenous VWF to FVIII. In a particular embodiment, the VWF fragment is not amino acids 764 to 1274 of SEQ ID NO: 2.

In some embodiments, a VWF fragment of the invention comprises the D' domain and the D3 domain, but does not comprise at least one VWF domain selected from the group consisting of (1) an Al domain, (2) an A2 domain, (3) an A3 domain, (4) a D4 domain, (5) a BI domain, (6) a B2 domain, (7) a B3 domain, (8) a Cl domain, (9) a C2 domain, (10) a CK domain, (11) a CK domain and C2 domain, (12) a CK domain, a C2 domain, and a Cl domain, (13) a CK domain, a C2 domain, a Cl domain, a B3 domain, (14) a CK domain, a C2 domain, a Cl domain, a B3 domain, a B2 domain, (15) a CK domain, a C2 domain, a Cl

-742018203206 08 May 2018 [0150] [0151] [0152] domain, a B3 domain, a B2 domain, and a BI domain, (16) a CK domain, a C2 domain, a Cl domain, a B3 domain, a B2 domain, a BI domain, and a D4 domain, (17) a CK domain, a C2 domain, a Cl domain, a B3 domain, a B2 domain, a BI domain, a D4 domain, and an A3 domain, (18) a CK domain, a C2 domain, a Cl domain, a B3 domain, a B2 domain, a BI domain, a D4 domain, an A3 domain, and an A2 domain, (19) a CK domain, a C2 domain, a Cl domain, a B3 domain, a B2 domain, a BI domain, a D4 domain, an A3 domain, an A2 domain, and an Al domain, and (20) any combinations thereof.

In yet other embodiments, the VWF fragment comprises the D'D3 domains and one or more domains or modules. Examples of such domains or modules include, but are not limited to, the domains and modules disclosed in Zhour et al., Blood published online April 6, 2012: DOI 10.1182/blood-2012-01-405134. For example, the VWF fragment can comprise the D'D3 domain and one or more domains or modules selected from the group consisting of Al domain, A2 domain, A3 domain, D4N module, VWD4 module, C8-4 module, TIL-4 module, Cl module, C2 module, C3 module, C4 module, C5 module, C5 module, C6 module, and any combinations thereof.

In still other embodiments, the VWF fragment is linked to a heterologous moiety, wherein the heterologous moiety is linked to the N-terminus or the Cterminus of the VWF fragment or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein in the VWF fragment. For example, the insertion sites for the heterologous moiety in the VWF fragment can be in the D' domain, the D3 domain, or both. The heterologous moiety can be a half-life extender.

In certain embodiments, a VWF fragment of the invention forms a multimer, e.g., dimer, trimer, tetramer, pentamer, hexamer, heptamer, or the higher order multimers. In other embodiments, the VWF fragment is a monomer having only one VWF fragment. In some embodiments, the VWF fragment of the present invention can have one or more amino acid substitutions, deletions, additions, or modifications. In one embodiment, the VWF fragment can include amino acid substitutions, deletions, additions, or modifications such that the VWF fragment is not capable of forming a disulfide bond or forming a dimer or a multimer. In another embodiment, the amino acid substitution is within the D'

-75 2018203206 08 May 2018 [0153] [0154] domain and the D3 domain. In a particular embodiment, a VWF fragment of the invention contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. The at least one amino acid substitution can be any amino acids that are not occurring naturally in the wild type VWF. For example, the amino acid substitution can be any amino acids other than cysteine, e.g., Isoleucine, Alanine, Leucine, Asparagine, Lysine, Aspartic acid, Methionine, Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine, Arginine, or Histidine. In another example, the amino acid substitution has one or more amino acids that prevent or inhibit the VWF fragments from forming multimers.

In certain embodiments, the VWF fragment useful herein can be further modified to improve its interaction with FVIII, e.g., to improve binding affinity to FVIII. As a non-limiting example, the VWF fragment comprises a serine residue at the residue corresponding to amino acid 764 of SEQ ID NO: 2 and a lysine residue at the residue corresponding to amino acid 773 of SEQ ID NO: 2. Residues 764 and/or 773 can contribute to the binding affinity of the VWF fragments to FVIII. In other embodiments, the VWF fragments useful for the invention can have other modifications, e.g., the protein can be pegylated, glycosylated, hesylated, or polysialylated.

B) XTEN Sequences

As used here XTEN sequence refers to extended length polypeptides with non-naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. As a chimeric protein partner, XTENs can serve as a carrier, conferring certain desirable pharmacokinetic, physicochemical and pharmaceutical properties when linked to a VWF fragment or a FVIII sequence of the invention to create a chimeric protein. Such desirable properties include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics. As used herein, XTEN specifically excludes antibodies or antibody fragments such as single-chain antibodies or Fc fragments of a light chain or a heavy chain.

-762018203206 08 May 2018 [0155] [0156] [0157]

In some embodiments, the XTEN sequence of the invention is a peptide or a polypeptide having greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, XTEN is a peptide or a polypeptide having greater than about 20 to about 3000 amino acid residues, greater than 30 to about 2500 residues, greater than 40 to about 2000 residues, greater than 50 to about 1500 residues, greater than 60 to about 1000 residues, greater than 70 to about 900 residues, greater than 80 to about 800 residues, greater than 90 to about 700 residues, greater than 100 to about 600 residues, greater than 110 to about 500 residues, or greater than 120 to about 400 residues.

The XTEN sequence of the invention can comprise one or more sequence motif of 9 to 14 amino acid residues or an amino acid sequence at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, wherein the motif comprises, consists essentially of, or consists of 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). See US 20100239554 Al.

In some embodiments, the XTEN comprises non-overlapping sequence motifs in which about 80%, or at least about 85%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% or about 100% of the sequence consists of multiple units of non-overlapping sequences selected from a single motif family selected from Table 2A, resulting in a family sequence. As used herein, family means that the XTEN has motifs selected only from a single motif category from Table 2A; i.e., AD, AE, AF, AG, AM, AQ, BC, or BD XTEN, and that any other amino acids in the XTEN not from a family motif are selected to achieve a needed property, such as to permit incorporation of a restriction site by the encoding nucleotides, incorporation of a cleavage sequence, or to achieve a better linkage to FVIII or VWF. In some embodiments of XTEN families, an XTEN sequence comprises multiple units of non-overlapping sequence motifs of the AD motif family, or of the AE motif family, or of the AF motif family, or of the AG motif family, or of the AM motif family, or of the AQ motif family, or of the BC family,

-772018203206 08 May 2018 or of the BD family, with the resulting XTEN exhibiting the range of homology described above. In other embodiments, the XTEN comprises multiple units of motif sequences from two or more of the motif families of Table 2 A. These sequences can be selected to achieve desired physical/chemical characteristics, including such properties as net charge, hydrophilicity, lack of secondary structure, or lack of repetitiveness that are conferred by the amino acid composition of the motifs, described more fully below. In the embodiments hereinabove described in this paragraph, the motifs incorporated into the XTEN can be selected and assembled using the methods described herein to achieve an XTEN of about 36 to about 3000 amino acid residues.

Table 2A. XTEN Sequence Motifs of 12 Amino Acids and Motif Families

Motif Family	MOTIF SEQUENCE
AD	GESPGGSSGSES
AD	GSEGSSGPGESS
AD	GSSESGSSEGGP
AD	GSGGEPSESGSS
AE, AM	GSPAGSPTSTEE
AE, AM, AQ	GSEPATSGSETP
AE, AM, AQ	GTSESATPESGP
AE, AM, AQ	GTSTEPSEGSAP
AF, AM	GSTSESPSGTAP
AF, AM	GTSTPESGSASP
AF, AM	GTSPSGESSTAP
AF, AM	GSTSSTAESPGP
AG, AM	GTPGSGTASSSP
AG, AM	GSSTPSGATGSP
AG, AM	GSSPSASTGTGP
AG, AM	GASPGTSSTGSP
AQ	GEPAGSPTSTSE
AQ	GTGEPSSTPASE
AQ	GSGPSTESAPTE
AQ	GSETPSGPSETA
AQ	GPSETSTSEPGA
AQ	GSPSEPTEGTSA
BC	GSGASEPTSTEP
BC	GSEPATSGTEPS
BC	GTSEPSTSEPGA
BC	GTSTEPSEPGSA
BD	GSTAGSETSTEA
BD	GSETATSGSETA
BD	GTSESATSESGA
BD	GTSTEASEGSAS

-782018203206 08 May 2018 [0158] [0159] • Denotes individual motif sequences that, when used together in various permutations, results in a family sequence

XTEN can have varying lengths for insertion into or linkage to FVIII or VWF. In one embodiment, the length of the XTEN sequence(s) is chosen based on the property or function to be achieved in the fusion protein. Depending on the intended property or function, XTEN can be short or intermediate length sequence or longer sequence that can serve as carriers. In certain embodiments, the XTEN include short segments of about 6 to about 99 amino acid residues, intermediate lengths of about 100 to about 399 amino acid residues, and longer lengths of about 400 to about 1000 and up to about 3000 amino acid residues. Thus, the XTEN inserted into or linked to FVIII or VWF can have lengths of about 6, about 12, about 36, about 40, about 42, about 72, about 96, about 144, about 288, about 400, about 500, about 576, about 600, about 700, about 800, about 864, about 900, about 1000, about 1500, about 2000, about 2500, or up to about 3000 amino acid residues in length. In other embodiments, the XTEN sequences is about 6 to about 50, about 50 to about 100, about 100 to 150, about 150 to 250, about 250 to 400, about 400 to about 500, about 500 to about 900, about 900 to 1500, about 1500 to 2000, or about 2000 to about 3000 amino acid residues in length. The precise length of an XTEN inserted into or linked to FVIII or VWF can vary without adversely affecting the activity of the FVIII or VWF. In one embodiment, one or more of the XTEN used herein has 36 amino acids, 42 amino acids, 72 amino acids, 144 amino acids, 288 amino acids, 576 amino acids, or 864 amino acids in length and can be selected from one or more of the XTEN family sequences; i.e., AD, AE, AF, AG, AM, AQ, BC or BD.

In some embodiments, the XTEN sequence used in the invention is at least

60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of AE42, AG42, AE48, AM48, AE72, AG72, AE108, AG108, AE144, AF144, AG144, AE180, AG180, AE216, AG216, AE252, AG252, AE288, AG288, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AG468, AE504, AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864,

-792018203206 08 May 2018 [0160] [0161] [0162]

AG864, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044,

ΑΕΙ 140, AE1236, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812,

AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428,

AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. See US 20100239554 Al.

In one embodiment, the XTEN sequence is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144 2A (SEQ IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO: 130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 39), AE288 2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG 144 (SEQ ID NO: 38), and any combinations thereof.

In some embodiments, less than 100% of amino acids of an XTEN are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consists of the sequence motifs from Table 2 A or the XTEN sequences of Table 2B. In such embodiments, the remaining amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but may be preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The content of hydrophobic amino acids in the XTEN utilized in the conjugation constructs may be less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, XTEN sequences may contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: methionine (for example, to avoid oxidation), or asparagine and glutamine (to avoid desamidation).

In another embodiment, the XTEN sequence is selected from the group consisting of AE42 (SEQ ID NO: 36), AE72 (SEQ ID NO: 127), AE144 2A

-802018203206 08 May 2018 (SEQ IDNO: 128), AE144 3B (SEQ ID NO: 129), AE144 4A (SEQ ID NO:

130), AE144 5A (SEQ IDNO: 131), AE144 6B (SEQ IDNO: 132), AG144 A (SEQ ID NO: 133), AG144 B (SEQ IDNO: 134), AG144 C (SEQ ID NO: 135), AG144 F (SEQ IDNO: 136), AE864 (SEQ ID NO: 43), AE576 (SEQ ID NO: 41), AE288 (SEQ IDNO: 39), AE288 2 (SEQ ID NO: 137), AE144 (SEQ ID NO: 37), AG864 (SEQ ID NO: 44), AG576 (SEQ ID NO: 42), AG288 (SEQ ID NO: 40), AG 144 (SEQ ID NO: 38), and any combinations thereof. In a specific embodiment, the XTEN sequence is AE288. The amino acid sequences for certain XTEN sequences of the invention are shown in Table 2B.

TABLE 2B. XTEN Sequences

XTEN	Amino Acid Sequence
AE42 SEQ ID NO: 36	GAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPASS
AE72	GAPTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA
SEQ ID NO: 127	TSGSETPGTSESATPESGPGTSTEPSEGSAPGASS
AE144	GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTE
SEQ ID	EGTSTEPSEG
NO:37	SAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESA PESGPGSEPATSGSETPGTSTEPSEGSAP
AE144 2A	TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP
(SEQ ID NO:	GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET
PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPES
128)	GPG
AE144 3B	SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
(SEQ ID NO:	GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
129)	APG
AE144 4A	TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
(SEQ ID NO:	GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS
130)	APG
AE144 5A	TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
(SEQ ID NO:	GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG
PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST

-81 2018203206 08 May 2018

131)	EEG
AE144 6B (SEQ ID NO: 132)	TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APG
AG 144 SEQ ID NO:38	GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTG PGASPGTSST GSPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSS TGSPGSSPSA STGTGPGTPGSGTASSSPGSSTPSGATGSP
AG144_A (SEQ ID NO: 133)	GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGASPGTSSTGSPGASPGTSST GSP
AG144B (SEQ ID NO: 134)	GTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSS PGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST GSP
AG144_C (SEQ ID NO: 135)	GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGS PGSSPSASTGTGPGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTG SPGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP
AG144F (SEQ ID NO: 136)	GSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSST GSP
AE288 SEQ ID NO:39	GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAP
AE288_2 (SEQ ID NO: 137)	GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAP
AG288	PGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG

-822018203206 08 May 2018

SEQ NO:40	ID	SPGTPGSGTASS SPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGAT GSPGSSPSASTG TGPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGA TGSPGSSPSAST GTGPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSPSAS TGTGPGASPGTSSTGSPGSSPSASTGTGPGTPGSGTASSSPGSSTPS GATGS
AE576 SEQ	ID	GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE
NO:41		TPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
AG576 SEQ	ID	PGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGT GPGSSTPSGATG SPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSST
NO:42		GSPGTPGSGTAS SSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSAST GTGPGTPGSGTA SSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSG ATGSPGSSTPSG ATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPS GATGSPGSSTPS GATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGS GTASSSPGASPG TSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG SGTASSSPGSST PSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSS TPSGATGSPGSS TPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSPGT PGSGTASSSPGS STPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS
AE864		GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSA

-832018203206 08 May 2018

SEQ NO:43	ID	PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTST EEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSG SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT PESGPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSP AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
AG864		GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSS
SEQ	ID	PGSSTPSGATGS PGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATG
NO:44		SPGTPGSGTASS SPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGAT GSPGASPGTSST GSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSAST GTGPGSSTPSGA TGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTS STGSPGTPGSGT ASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSA STGTGPGTPGSG TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTP SGATGSPGSSTP SGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSST PSGATGSPGSST PSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTP GSGTASSSPGAS PGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT PGSGTASSSPGS STPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPG SSTPSGATGSPG SSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP GTPGSGTASSSP

-842018203206 08 May 2018

GSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGS PGASPGTSSTGS PGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSASTGT GPGTPGSGTASS SPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP

[0163] In further embodiments, the XTEN sequence used in the invention affects the physical or chemical property, e.g., pharmacokinetics, of the chimeric protein of the present invention. The XTEN sequence used in the present invention can exhibit one or more of the following advantageous properties: conformational flexibility, enhanced aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radii. In a specific embodiment, the XTEN sequence linked to a FVIII protein in this invention increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that the chimeric protein described herein stays in vivo for an increased period of time compared to wild type FVIII. In further embodiments, the XTEN sequence used in this invention increases pharmacokinetic properties such as longer terminal half-life or increased area under the curve (AUC), so that FVIII protein stays in vivo for an increased period of time compared to wild type FVIII.

[0164] A variety of methods and assays can be employed to determine the physical/chemical properties of proteins comprising the XTEN sequence. Such methods include, but are not limited to analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractometry, and UV/Visible spectroscopy. Additional methods are disclosed in Amau et al., Prot Expr and Purif 48, 1-13 (2006).

[0165] Additional examples of XTEN sequences that can be used according to the present invention and are disclosed in US Patent Publication Nos. 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, or 2011/0172146 Al, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2.

C) Factor VIII (FVIII) Protein

-852018203206 08 May 2018 [0166] [0167] [0168] [0169]

A FVIII protein as used herein means a functional FVIII polypeptide in its normal role in coagulation, unless otherwise specified. The term a FVIII protein includes a functional fragment, variant, analog, or derivative thereof that retains the function of full-length wild-type Factor VIII in the coagulation pathway. A FVIII protein is used interchangeably with FVIII polypeptide (or protein) or FVIII. Examples of the FVIII functions include, but not limited to, an ability to activate coagulation, an ability to act as a cofactor for factor IX, or an ability to form a tenase complex with factor IX in the presence of Ca and phospholipids, which then converts Factor X to the activated form Xa. The FVIII protein can be the human, porcine, canine, rat, or murine FVIII protein. In addition, comparisons between FVIII from humans and other species have identified conserved residues that are likely to be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); US 6,251,632).

A number of tests are available to assess the function of the coagulation system: activated partial thromboplastin time (aPTT) test, chromogenic assay, ROTEM assay, prothrombin time (PT) test (also used to determine INR), fibrinogen testing (often by the Clauss method), platelet count, platelet function testing (often by PFA-100), TCT, bleeding time, mixing test (whether an abnormality corrects if the patient's plasma is mixed with normal plasma), coagulation factor assays, antiphospholipid antibodies, D-dimer, genetic tests (e.g., factor V Leiden, prothrombin mutation G20210A), dilute Russell's viper venom time (dRVVT), miscellaneous platelet function tests, thromboelastography (TEG or Sonoclot), thromboelastometry (TEM®, e.g., ROTEM®), or euglobulin lysis time (ELT).

The aPTT test is a performance indicator measuring the efficacy of both the intrinsic (also referred to the contact activation pathway) and the common coagulation pathways. This test is commonly used to measure clotting activity of commercially available recombinant clotting factors, e.g., FVIII or FIX. It is used in conjunction with prothrombin time (PT), which measures the extrinsic pathway.

ROTEM analysis provides information on the whole kinetics of haemostasis: clotting time, clot formation, clot stability and lysis. The different parameters in thromboelastometry are dependent on the activity of the plasmatic coagulation system, platelet function, fibrinolysis, or many factors which

-86influence these interactions. This assay can provide a complete view of secondary haemostasis.

The FVIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified versions. Examples of human

FVIII sequences (full-length) are shown below.

TABLE 3. Amino Acid Sequence of Full-length Factor VIII (Full-length FVIII (FVIII signal peptide underlined; FVIII heavy chain is double underlined; B domain is italicized; and FVIII light chain is in plain text)

2018203206 08 May 2018 [0170]

Signal Peptide: (SEQ ID NO: 3)

MQIELSTCFFLCLLRFCFS

Mature Factor VIII (SEQ ID NO: 4)*

ATRRYYLGAVELSWPYMQSDLGELPVPARFPPRVPKSFPFNTSVVYKKTLFVEFT

DHLFNIAKPRPPWMGLLGPTTQAEVYDTVViTLKNMASHPVSLHAVGVSYWKASE

GAEYDpyrSQI^KEDDKVFPGGSHTYWQVLKENGPMAS'DPLCLTYSYLSHVDLAZ

KPLRSGLIGALLVCREGSLAKEKTQTLHKF'ILLF'AVFPEGKSWHSETKNSLMQPR

DAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHT

FLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCP

EEPQLRMKNNEEAEPYPPPLTPSEMPVVRFPPPNSPSFIQIRSVAKKHPKTWVHY

TAAEEEPWPYAPLVLAPPPRSYKSQYLNNGPQRIGRKYKKVRFMAYTPETFKTRE

AIQHESGTLGPLLYGE^DTLLTTi^NQASRPYNlYPHGTTDVRPLYSRRLPKGV

KHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP

LLICYKESVPQRGNQIMSPKRNVILFSVFPENRSWYLTENIQRFLPNPAGVQLEP

PEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKH

KMVYEPTLTLFPFSGETVFMSMENPGLWILGCHNSPFRNRGMTALLKVSSCPKNT

GDY YE PS YE DISAYLL SKNNAIE PRSFSGNSR.S.PSIROFsFIS^IIIFENBPEKTE PEEAEEPPEiPKIGEVSSSDEEPiLEPOSPIPEGESLSDEQE-AEAFIFSEDPSPGAP DSNNSLSPPiPPFEPQPEESGDMVFIPPSGLQPELNPKLGIIAAIELEKLDEKVSS ISNNLPSIIPSDNLAAGPDNISSPGPPSP;PVHYDSGLlG^:ILFGKKSSPLPPSGGP LSPSEENNDS.KPLEGGLIiPSQEGSEG.KEVSGIESGELEKGKPAHGPAPLPKDNAP EEVSPGPPKTNKPGNNSAINREIEPDGPSEIIENSPSyWGNPPESDIEEEKyppP PFPEFilPDKNATAIJlFEFPSNEIEFSKNEFIA/QQKKEGPIFPPAQPPDFiSFFKEP EL PE SAP FI QF FEGKES.LNSGQGPS PKOL VSLGPEKS VEGQNPL SEKEKV VVGE G EFIKDVSLKPPr/EPSSPNLRL lELDNLEENEPPNQEKKIQEPIEKKEIL IQENVV EPQLNTVPGTKEENKEEFLLSTEQNVEGSYDGAVAPVlQVNIISLNDSPNPIKKFI AKFS.KKGEPEEIPGLGNQIEGKYP.KYACIPRPSPEISQQEEVIQPSKPALKQLKL PEEEPELE.KEPPVDDEGTQGGKNEKELEPSPLPGPDYNEEEKGAITQSPESDGEP PSKG IPQA.NESPPPPA.KySG FPSIPPPII IRFI.FQ PFSSHIPAAS YRFIFSG POP SSFFFOGAKKNNFSLA IL PEEPIEGDOPEVGSEGISA INS VIYKEVENIVLF.KPEL PKPSGKVEPIMEVEIYGKDIFPPFIENGSPSFPELVEGSIPOSIEGAPKENEAKP PGKVPELPVALFSSAKIPSKLLDPLAEDEEYGIQIPKEFGKSQFKSPFKIAFKKK DIIININACFSNEAIAAINFGQNKFFIFVINAKQGPIERLCSGKFPVLKPHQKEI TRTTLQSPQEEIPYPPTISVEMKKEPFPIYPEPENQSPRSFQKKTRHYFIAAVER LWPYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTPGSFTQPLYRGELNEHLGLLG

-872018203206 08 May 2018

PYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTY FWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQ VTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYI MDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYP GVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDF QITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGAR QKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMF ATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSM YVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHP QSWVHQIALRMEVLGCEAQDLY

TABLE 4. Nucleotide Sequence Encoding Full-Length FVIII (SEQ ID NO: 5)*

661 ATG

CAAATAGAGC TCTCCACCTG

721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC

781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG

841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCAGTCGTGT ACAAAAAGAC

901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT

961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA

1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC

1081 TGAGGGAGCT GAATATGATG ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT

1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC

1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA

1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC

1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA

1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC

1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA

1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT

2018203206 08 May 2018

1561 ATTCCTCGAA GGTCACACAT CAGGCGTCCT TGGAAATCTC

1621 GCCAATAACT TTCCTTACTG CTTGGACAGT TTCTACTGTT

1681 TTGTCATATC TCTTCCCACC TATGTCAAAG TAGACAGCTG 1741 TCCAGAGGAA CCCCAACTAC

GCGGAAGACT ATGATGATGA 1801 TCTTACTGAT TCTGAAATGG

GACAACTCTC CTTCCTTTAT 1861 CCAAATTCGC TCAGTTGCCA

GTACATTACA TTGCTGCTGA 1921 AGAGGAGGAC TGGGACTATG

GATGACAGAA GTTATAAAAG 1981 TCAATATTTG AACAATGGCC

TACAAAAAAG TCCGATTTAT 2041 GGCATACACA GATGAAACCT

CAGCATGAAT CAGGAATCTT 2101 GGGACCTTTA CTTTATGGGG

ATTATATTTA AGAATCAAGC 2161 AAGCAGACCA TATAACATCT

GTCCGTCCTT TGTATTCAAG 2221 GAGATTACCA AAAGGTGTAA

ATTCTGCCAG GAGAAATATT 2281 CAAATATAAA TGGACAGTGA

AAATCAGATC CTCGGTGCCT 2341 GACCCGCTAT TACTCTAGTT

CTAGCTTCAG GACTCATTGG 2401 CCCTCTCCTC ATCTGCTACA

GGAAACCAGA TAATGTCAGA 2461 CAAGAGGAAT GTCATCCTGT

CGAAGCTGGT ACCTCACAGA 2521 GAATATACAA CGCTTTCTCC

CTTGAGGATC CAGAGTTCCA 2581 AGCCTCCAAC ATCATGCACA

GATAGTTTGC AGTTGTCAGT 2641 TTGTTTGCAT GAGGTGGCAT

GGAGCACAGA CTGACTTCCT 2701 TTCTGTCTTC TTCTCTGGAT

GTCTATGAAG ACACACTCAC 2761 CCTATTCCCA TTCTCAGGAG

GAAAACCCAG GTCTATGGAT 2821 TCTGGGGTGC CACAACTCAG

ACCGCCTTAC TGAAGGTTTC 2881 TAGTTGTGAC AAGAACACTG

TATGAAGATA TTTCAGCATA 2941 CTTGCTGAGT AAAAACAATG TCCCAGAATT CAAGACACCC 3001 TAGCACTAGG CAAAAGCAAT

GAAAATGACA TAGAGAAGAC

TTCTTGTGAG GAACCATCGC CTCAAACACT CTTGATGGAC AACATGATGG CATGGAAGCT
GAATGAAAAA	TAATGAAGAA
ATGTGGTCAG	GTTTGATGAT
AGAAGCATCC	TAAAACTTGG
CTCCCTTAGT	CCTCGCCCCC
CTCAGCGGAT	TGGTAGGAAG
TTAAGACTCG	TGAAGCTATT
AAGTTGGAGA	CACACTGTTG
ACCCTCACGG	AATCACTGAT
AACATTTGAA	GGATTTTCCA
CTGTAGAAGA	TGGGCCAACT
TCGTTAATAT	GGAGAGAGAT
AAGAATCTGT	AGATCAAAGA
TTTCTGTATT	TGATGAGAAC
CCAATCCAGC	TGGAGTGCAG
GCATCAATGG	CTATGTTTTT
ACTGGTACAT	TCTAAGCATT
ATACCTTCAA	ACACAAAATG
AAACTGTCTT	CATGTCGATG
ACTTTCGGAA	CAGAGGCATG
GTGATTATTA	CGAGGACAGT
CCATTGAACC	AAGAAGCTTC
TTAATGCCAC	CACAATTCCA

2018203206 08 May 2018

3061 TGACCCTTGG TTTGCACACA CAAAATGTCT CCTCTAGTGA

3121 TTTGTTGATG CTCTTGCGAC CTATCCTTAT CTGATCTCCA

3181 AGAAGCCAAA TATGAGACTT GGAGCAATAG ACAGTAATAA

3241 CAGCCTGTCT GAAATGACAC CACAGTGGGG ACATGGTATT

3301 TACCCCTGAG TCAGGCCTCC CTGGGGACAA CTGCAGCAAC

3361 AGAGTTGAAG AAACTTGATT AATAATCTGA TTTCAACAAT

3421 TCCATCAGAC AATTTGGCAG TCCTTAGGAC CCCCAAGTAT

3481 GCCAGTTCAT TATGATAGTC GGCAAAAAGT CATCTCCCCT

3541 TACTGAGTCT GGTGGACCTC AATGATTCAA AGTTGTTAGA

3601 ATCAGGTTTA ATGAATAGCC AATGTATCGT CAACAGAGAG

3661 TGGTAGGTTA TTTAAAGGGA TTGTTGACTA AAGATAATGC

3721 CTTATTCAAA GTTAGCATCT ACTTCCAATA ATTCAGCAAC

3781 TAATAGAAAG ACTCACATTG GAGAATAGTC CATCAGTCTG

3841 GCAAAATATA TTAGAAAGTG ACACCTTTGA TTCATGACAG

3901 AATGCTTATG GACAAAAATG CATATGTCAA ATAAAACTAC

3961 TTCATCAAAA AACATGGAAA GGCCCCATTC CACCAGATGC

4021 ACAAAATCCA GATATGTCGT CCAGAATCAG CAAGGTGGAT

4081 ACAAAGGACT CATGGAAAGA GGCCCCAGTC CAAAGCAATT

4141 AGTATCCTTA GGACCAGAAA TTCTTGTCTG AGAAAAACAA

4201 AGTGGTAGTA GGAAAGGGTG CTCAAAGAGA TGGTTTTTCC

4261 AAGCAGCAGA AACCTATTTC CATGAAAATA ATACACACAA

3 21 TCAAGAAAAA AAAAT TCAGG ACATTAATCC AAGAGAATGT

4381 AGTTTTGCCT CAGATACATA TTCATGAAGA ACCTTTTCTT

4441 ACTGAGCACT AGGCAAAATG GCATATGCTC CAGTACTTCA

4501 AGATTTTAGG TCATTAAATG AAACACACAG CTCATTTCTC

GAACACCTAT

AGAGTCCTAC

TTTCTGATGA

ACTTCAGGCC

AATTAAGATT

TCAAAGTTTC

CAGGTACTGA

AATTAGATAC

TGAGCTTGAG

AAGAAAGTTC

AAAGAGCTCA

CTTTGTTAAA

ATGGCCCATC

ACACTGAGTT

CTACAGCTTT

TGGTCCAACA

TCTTTAAGAT

ACTCTCTGAA

AATCTGTGGA

AATTTACAAA

TTACTAACTT

AAGAAATAGA

CAGTGACTGG

TAGAAGGTTC

ATTCAACAAA

GCCTAAAATA

TCCACATGGG

TCCATCACCT

ACAGCTCCAT

AAATGAGAAA

TAGTACATCA

TAATACAAGT

CACTCTATTT

TGAAGAAAAT

ATGGGGAAAA

TGGACCTGCT

GACAAACAAA

ATTATTAATT

TAAAAAAGTG

GAGGCTAAAT

GAAAAAAGAG

GCTATTCTTG

CTCTGGGCAA

AGGTCAGAAT

GGACGTAGGA

GGATAATTTA

AAAGAAGGAA

CACTAAGAAT

ATATGACGGG

TAGAACAAAG

-902018203206 08 May 2018

4561 AAAAAAAGGG GAGGAAGAAA CAAACCAAGC AAATTGTAGA

4621 GAAATATGCA TGCACCACAA CAGCAGAATT TTGTCACGCA

4681 ACGTAGTAAG AGAGCTTTGA GAAGAAACAG AACTTGAAAA

4741 AAGGATAATT GTGGATGACA AACATGAAAC ATTTGACCCC

4801 GAGCACCCTC ACACAGATAG GGGGCCATTA CTCAGTCTCC

4861 CTTATCAGAT TGCCTTACGA GCAAATAGAT CTCCATTACC

4921 CATTGCAAAG GTATCATCAT TATCTGACCA GGGTCCTATT

4981 CCAAGACAAC TCTTCTCATC AAGAAAGATT CTGGGGTCCA

5041 AGAAAGCAGT CATTTCTTAC CTTTCTTTAG CCATTCTAAC

5101 CTTGGAGATG ACTGGTGATC GGGACAAGTG CCACAAATTC

5161 AGTCACATAC AAGAAAGTTG CCAGACTTGC CCAAAACATC

5221 TGGCAAAGTT GAATTGCTTC AAGGACCTAT TCCCTACGGA

5281 AACTAGCAAT GGGTCTCCTG GGGAGCCTTC TTCAGGGAAC

5341 AGAGGGAGCG ATTAAGTGGA AAAGTTCCCT TTCTGAGAGT

5401 AGCAACAGAA AGCTCTGCAA GATCCTCTTG CTTGGGATAA

5461 CCACTATGGT ACTCAGATAC CAAGAGAAGT CACCAGAAAA

5 21 AACAGCT TTT AAGAAAAAGG GCTTGTGAAA GCAATCATGC

5581 AATAGCAGCA ATAAATGAGG GAAGTCACCT GGGCAAAGCA

5641 AGGTAGGACT GAAAGGCTGT TTGAAACGCC ATCAACGGGA

5701 AATAACTCGT ACTACTCTTC GACTATGATG ATACCATATC

5761 AGTTGAAATG AAGAAGGAAG GATGAAAATC AGAGCCCCCG

8 21 CAGCTTTCAA AAGAAAACAC GTGGAGAGGC TCTGGGATTA

5881 TGGGATGAGT AGCTCCCCAC CAGAGTGGCA GTGTCCCTCA

5941 GTTCAAGAAA GTTGTTTTCC TTTACTCAGC CCTTATACCG

6001 TGGAGAACTA AATGAACATT ATAAGAGCAG AAGTTGAAGA

ACTTGGAAGG

GGATATCTCC

AACAATTCAG

CCTCAACCCA

ACTACAATGA

GGAGTCATAG

TTCCATCTAT

TTCCAGCAGC

AAGGAGCCAA

AAAGAGAGGT

AGAACACTGT

CAAAAGTTCA

GCCATCTGGA

ATGAAGCAAA

AGACTCCCTC

CAAAAGAAGA

ATACCATTTT

GACAAAATAA

GCTCTCAAAA

AGTCAGATCA

ATTTTGACAT

GACACTATTT

ATGTTCTAAG

AGGAATTTAC

TGGGACTCCT

CTTGGGAAAT

TAATACAAGC

ACTCCCACTA

GTGGTCCAAA

GAAGGAGAAA

CATCCCTCAA

TAGACCTATA

ATCTTATAGA

AAAAAATAAC

TGGCTCCCTG

TCTCCCGAAA

CATTTATCAG

TCTCGTGGAA

CAGACCTGGA

CAAGCTATTG

GTGGAAATCC

GTCCCTGAAC

GCCCGAAATA

CCCACCAGTC

AGAGGAAATT

TTATGATGAG

TATTGCTGCA

AAACAGGGCT

TGATGGCTCC

GGGGCCATAT

2018203206 08 May 2018

6061 TAATATCATG GTAACTTTCA TATTCCTTCT ATTCTAGCCT

6121 TATTTCTTAT GAGGAAGATC AGAAAAAACT TTGTCAAGCC

6181 TAATGAAACC AAAACTTACT ATGGCACCCA CTAAAGATGA

6241 GTTTGACTGC AAAGCCTGGG CTGGAAAAAG ATGTGCACTC

6301 AGGCCTGATT GGACCCCTTC CTGAACCCTG CTCATGGGAG

6361 ACAAGTGACA GTACAGGAAT TTTGATGAGA CCAAAAGCTG

6421 GTACTTCACT GAAAATATGG TGCAATATCC AGATGGAAGA

6481 TCCCACTTTT AAAGAGAATT GGCTACATAA TGGATACACT

6541 ACCTGGCTTA GTAATGGCTC TATCTGCTCA GCATGGGCAG

6601 CAATGAAAAC ATCCATTCTA TTCACTGTAC GAAAAAAAGA

6661 GGAGTATAAA ATGGCACTGT TTTGAGACAG TGGAAATGTT

6721 ACCATCCAAA GCTGGAATTT GGCGAGCATC TACATGCTGG

6781 GATGAGCACA CTTTTTCTGG ACTCCCCTGG GAATGGCTTC

6841 TGGACACATT AGAGATTTTC TATGGACAGT GGGCCCCAAA

6901 GCTGGCCAGA CTTCATTATT AGCACCAAGG AGCCCTTTTC

6961 TTGGATCAAG GTGGATCTGT GGCATCAAGA CCCAGGGTGC

7021 CCGTCAGAAG TTCTCCAGCC ATCATGTATA GTCTTGATGG

7081 GAAGAAGTGG CAGACTTATC TTAATGGTCT TCTTTGGCAA

7141 TGTGGATTCA TCTGGGATAA CCAATTATTG CTCGATACAT

7201 CCGTTTGCAC CCAACTCATT CGCATGGAGT TGATGGGCTG

7261 TGATTTAAAT AGTTGCAGCA AAAGCAATAT CAGATGCACA

7321 GATTACTGCT TCATCCTACT TGGTCTCCTT CAAAAGCTCG

7381 ACTTCACCTC CAAGGGAGGA GTGAATAATC CAAAAGAGTG

7441 GCTGCAAGTG GACTTCCAGA GTAACTACTC AGGGAGTAAA

7501 ATCTCTGCTT ACCAGCATGT TCCAGCAGTC AAGATGGCCA

GAAATCAGGC	CTCTCGTCCC
AGAGGCAAGG	AGCAGAACCT
TTTGGAAAGT	GCAACATCAT
CTTATTTCTC	TGATGTTGAC
TGGTCTGCCA	CACTAACACA
TTGCTCTGTT	TTTCACCATC
AAAGAAACTG	CAGGGCTCCC
ATCGCTTCCA	TGCAATCAAT
AGGATCAAAG	GATTCGATGG
TTCATTTCAG	TGGACATGTG
ACAATCTCTA	TCCAGGTGTT
GGCGGGTGGA	ATGCCTTATT
TGTACAGCAA	TAAGTGTCAG
AGATTACAGC	TTCAGGACAA
CCGGATCAAT	CAATGCCTGG
TGGCACCAAT	GATTATTCAC
TCTACATCTC	TCAGTTTATC
GAGGAAATTC	CACTGGAACC
AACACAATAT	TTTTAACCCT
ATAGCATTCG	CAGCACTCTT
TGCCATTGGG	AATGGAGAGT
TTACCAATAT	GTTTGCCACC
GTAATGCCTG	GAGACCTCAG
AGACAATGAA	AGTCACAGGA
ATGTGAAGGA	GTTCCTCATC

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7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT

TTTCAGGGAA ATCAAGACTC

7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG

ACTCGCTACC TTCGAATTCA

7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA

7741 GGACCTCTAC *The underlined nucleic acids encode a signal peptide.

[0171] FVIII polypeptides include lull-length FVIII, lull-length FVIII minus Met at the N-terminus, mature FVIII (minus the signal sequence), mature FVIII with an additional Met at the N-terminus, and/or FVIII with a lull or partial deletion of the B domain. In certain embodiments, FVIII variants include B domain deletions, whether partial or full deletions.

[0172] The sequence of native mature human FVIII is presented as SEQ ID NO:

4. A native FVIII protein has the following formula: Al-al-A2-a2-B-a3-A3-ClC2, where Al, A2, and A3 are the structurally-related A domains, B is the B domain, Cl and C2 are the structurally-related C domains, and al, a2 and a3 are acidic spacer regions. Referring to the primary amino acid sequence position in SEQ ID NO:4, the Al domain of human FVIII extends from Alai to about Arg336, the al spacer region extends from about Met337 to about Val374, the A2 domain extends from about Ala375 to about Tyr719, the a2 spacer region extends from about Glu720 to about Arg740, the B domain extends from about Ser741 to about Arg 1648, the a3 spacer region extends from about Glut649 to about Argl689, the A3 domain extends from about Seri690 to about Leu2025, the Cl domain extends from about Gly2026 to about Asn2072, and the C2 domain extends from about Ser2073 to Tyr2332. Other than specific proteolytic cleavage sites, designation of the locations of the boundaries between the domains and regions of FVIII can vary in different literature references. The boundaries noted herein are therefore designated as approximate by use of the term about.

[0173] The human FVIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337 (1984); Vehar, G. A., etal., Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; and U.S. Pat. No. 4,757,006). The FVIII amino acid sequence was

-93 2018203206 08 May 2018 [0174] [0175] [0176] deduced from cDNA as shown in U.S. Pat. No. 4,965,199. In addition, partially or fully B-domain deleted FVIII is shown in U.S. Pat. Nos. 4,994,371 and 4,868,112. In some embodiments, the human FVIII B-domain is replaced with the human Factor V B-domain as shown in U.S. Pat. No. 5,004,803. The cDNA sequence encoding human Factor VIII and amino acid sequence are shown in SEQ ID NOs: 4 and 5, respectively, of US Application Publ. No. 2005/0100990.

The porcine FVIII sequence is published in Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986). Further, the complete porcine cDNA sequence obtained from PCR amplification of FVIII sequences from a pig spleen cDNA library has been reported in Healey, J. F., et al., Blood 88:4209-4214 (1996). Hybrid human/porcine FVIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093. More recently, the nucleotide and corresponding amino acid sequences of the Al and A2 domains of porcine FVIII and a chimeric FVIII with porcine Al and/or A2 domains substituted for the corresponding human domains were reported in WO 94/11503. U.S. Pat. No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA and deduced amino acid sequences. U.S. Pat. No. 6,458,563 discloses a B-domaindeleted porcine FVIII.

U.S. Pat. No. 5,859,204 to Lollar, J. S. reports functional mutants of FVIII having reduced antigenicity and reduced immunoreactivity. U.S. Pat. No.

6,376,463 to Lollar, J. S. also reports mutants of FVIII having reduced immunoreactivity. US Appl. Publ. No. 2005/0100990 to Saenko et al. reports functional mutations in the A2 domain of FVIII.

In one embodiment, the FVIII (or FVIII portion of a chimeric protein) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence) or a FVIII amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 3 and 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 19 of SEQ ID NO: 3 and amino acids 1 to 2332 of SEQ ID NO: 4 (with a signal sequence), wherein the FVIII has a clotting activity, e.g., activates Factor IX as a cofactor to convert Factor X to activated Factor X. The FVIII (or FVIII portion of a chimeric protein) may be identical to a

-942018203206 08 May 2018

FVIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 6 or amino acids 1 to 2332 of SEQ ID NO: 4 (without a signal sequence). The FVIII may further comprise a signal sequence.

[0177] The B-domain of FVIII, as used herein, is the same as the B-domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741-Arg 1648 of full-length human FVIII. The other human FVIII domains are defined by the following amino acid residues: Al, residues Alal-Arg372; A2, residues Ser373-Arg740; A3, residues Seri690Asn2019; Cl, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Serl690-Tyr2332. The remaining sequence, residues Glul649-Argl689, is usually referred to as the a3 acidic region. The locations of the boundaries for all of the domains, including the B-domains, for porcine, mouse and canine FVIII are also known in the art. In one embodiment, the B domain of FVIII is deleted (B-domain-deleted factor VIII or BDD FVIII). An example of a BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequence as the Factor VIII portion of the sequence in Table 5. (BDD FVIII heavy chain is double underlined; B domain is italicized; and BDD FVIII light chain is in plain text). A nucleotide sequence encoding the amino acid sequence set forth in Table 5 (SEQ ID NO: 7) is shown in Table 6.

TABLE 5. Amino Acid Sequence of B-domain Deleted Factor VIII (BDD

FVIII)

BDD FVIII (SEQ ID NO: 6)

ATRRYYLGAyELSWDYMQSDLGELPyDARFPPRypKSFPFNTSyyYKKTLFyEFT

ΡΗΕΕΫΪΪΑΚΡ]ΫΡΕΜΡΚΕΪ^^

GAEYDDQTSOREKEDDKyFPGGSHTYyWOyLKENGPMASDPLCLTYSYLSHyDLy

KDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDR

DAASARAWPKMHTyNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEyHSIFLEGHT

FLyRNHRQASLETSPITFLTAQTLLMDLGQFLLFCHTSSHQHDGMEAYyKyDSCP

EEPQLRNKNNEEAEiGYDDDLTDSEMDWRFDDDNSPsHQTRSyAKKHPKTWyHY lAAEEEDWDYAPLyLAPDDRSYKSQYLENGPQRIGRKYKKyRFMjLYTDETFKTRE

AIOHESGILGPLLYGEyGbTLLIIFKNOASRPYNIYPHGITPyRPLYSRRLPKGy

KHLKDFPILPGEIFKYKWTyTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGP

LLICYKESyDQRGNQIMSDKRNyiLFSyFDENRSWYLTENiQRFLPNPAGyQLED

PEFQASNIMHSINGYVFDSLQLSyCLHEyAYWYILSIGAQTDFLSVFFSGYTFKH

KWYEDTLTLFPFSGETWMSMENPGLWTLGCHNSDroNRGRITALLKySSCDKNT

GDYYE DSYE DISAYLLSKNNAIE PRGFgOVRpyLFRPfORE Ϊ TRT T LQ S DQEE Ϊ DY

DDT Ϊ SVEMKKEDFDIYDEDENQS PRSFQKKTRHYFIAAVERLWDYGMS S S PHVLR

-95 2018203206 08 May 2018

NRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVT FRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDE FDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFD ETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAG IWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPK LARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIM YSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIR STLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQG RSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGH QWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY

TABLE 6. Nucleotide Sequence Encoding BDD FVIII (SEQ ID NO: 7)*

61 A TGCAAATAGA

GCTCTCCACC TGCTTCTTTC

721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC

781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC

841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG

901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC

961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG

1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG

1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG

1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGAGAATGG TCCAATGGCC TCTGACCCAC

1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC

1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT

1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA

1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC

1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT

1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG

1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA

1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA

-962018203206 08 May 2018

1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA

AGTAGACAGC TGTCCAGAGG

1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAAGA

CTATGATGAT GATCTTACTG

1801 ATTCTGAAAT GGATGTGGTC AGGTTTGATG ATGACAACTC TCCTTCCTTT ATCCAAATTC

1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG

1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT

1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA

2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT

2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC

2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC

2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA

2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT

2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC

2401 TCATCTGCTA CAAAGAATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA

2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC

2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA

2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC

2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT

2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC

2761 CATTCTCAGG AGAAACTGTC TTCATGTCGA TGGAAAACCC AGGTCTATGG ATTCTGGGGT

2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG

2881 ACAAGAACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA

2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC

3001 ATCAACGGGA AATAACTCGT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG

3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC

3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC

-972018203206 08 May 2018

3181 TCTGGGATTA TGGGATGAGT AAACAGGGCT CAGAGTGGCA

3241 GTGTCCCTCA GTTCAAGAAA TGATGGCTCC TTTACTCAGC

3301 CCTTATACCG TGGAGAACTA GGGGCCATAT ATAAGAGCAG

3361 AAGTTGAAGA TAATATCATG CTCTCGTCCC TATTCCTTCT

3421 ATTCTAGCCT TATTTCTTAT AGCAGAACCT AGAAAAAACT

3481 TTGTCAAGCC TAATGAAACC GCAACATCAT ATGGCACCCA

3541 CTAAAGATGA GTTTGACTGC TGATGTTGAC CTGGAAAAAG

3601 ATGTGCACTC AGGCCTGATT CACTAACACA CTGAACCCTG

3661 CTCATGGGAG ACAAGTGACA TTTCACCATC TTTGATGAGA

3721 CCAAAAGCTG GTACTTCACT CAGGGCTCCC TGCAATATCC

3781 AGATGGAAGA TCCCACTTTT TGCAATCAAT GGCTACATAA

3841 TGGATACACT ACCTGGCTTA GATTCGATGG TATCTGCTCA

3901 GCATGGGCAG CAATGAAAAC TGGACATGTG TTCACTGTAC

3961 GAAAAAAAGA GGAGTATAAA TCCAGGTGTT TTTGAGACAG

4021 TGGAAATGTT ACCATCCAAA ATGCCTTATT GGCGAGCATC

4081 TACATGCTGG GATGAGCACA TAAGTGTCAG ACTCCCCTGG

4141 GAATGGCTTC TGGACACATT TTCAGGACAA TATGGACAGT

4201 GGGCCCCAAA GCTGGCCAGA CAATGCCTGG AGCACCAAGG

4261 AGCCCTTTTC TTGGATCAAG GATTATTCAC GGCATCAAGA

4321 CCCAGGGTGC CCGTCAGAAG TCAGTTTATC ATCATGTATA

4381 GTCTTGATGG GAAGAAGTGG CACTGGAACC TTAATGGTCT

4441 TCTTTGGCAA TGTGGATTCA TTTTAACCCT CCAATTATTG

4501 CTCGATACAT CCGTTTGCAC CAGCACTCTT CGCATGGAGT

4561 TGATGGGCTG TGATTTAAAT AATGGAGAGT AAAGCAATAT

4621 CAGATGCACA GATTACTGCT GTTTGCCACC TGGTCTCCTT

AGCTCCCCAC	ATGTTCTAAG
GTTGTTTTCC	AGGAATTTAC
AATGAACATT	TGGGACTCCT
GTAACTTTCA	GAAATCAGGC
GAGGAAGATC	AGAGGCAAGG
AAAACTTACT	TTTGGAAAGT
AAAGCCTGGG	CTTATTTCTC
GGACCCCTTC	TGGTCTGCCA
GTACAGGAAT	TTGCTCTGTT
GAAAATATGG	AAAGAAACTG
AAAGAGAATT	ATCGCTTCCA
GTAATGGCTC	AGGATCAAAG
ATCCATTCTA	TTCATTTCAG
ATGGCACTGT	ACAATCTCTA
GCTGGAATTT	GGCGGGTGGA
CTTTTTCTGG	TGTACAGCAA
AGAGATTTTC	AGATTACAGC
CTTCATTATT	CCGGATCAAT
GTGGATCTGT	TGGCACCAAT
TTCTCCAGCC	TCTACATCTC
CAGACTTATC	GAGGAAATTC
TCTGGGATAA	AACACAATAT
CCAACTCATT	ATAGCATTCG
AGTTGCAGCA	TGCCATTGGG
TCATCCTACT	TTACCAATAT

2018203206 08 May 2018

/.

[0178] ±681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG

GAGACCTCAG GTGAATAATC ±741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA

AGTCACAGGA GTAACTACTC ±801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC ±861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ±921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC ±981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT ±1 GCGAGGCACA GGACCTCTAC *The underlined nucleic acids encode a signal peptide.

A B-domain-deleted FVIII may have the full or partial deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844,

5,112,950, 4,868,112, and 6,458,563. In some embodiments, a B-domain-deleted FVIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and Examples 1-5 of U.S. Pat. No. 6,316,226 (also in US 6,346,513). In another embodiment, a B-domain deleted Factor VIII is the S743/Q1638 B-domain deleted Factor VIII (SQ BDD FVIII) (e.g., Factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII having amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 4, i.e., SEQ ID NO: 6). In some embodiments, a B-domain-deleted FVIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Patent No. 5,789,203 (also US 6,060,447, US 5,595,886, and US 6,228,620). In some embodiments, a B-domain-deleted Factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of US Patent No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Patent no. 6,048,720; col. 2, lines 17-46 of U.S. Patent No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Patent no. 5,171,844; col. 2, lines 55-68, figure 2, and example 1 of U.S. Patent No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Patent No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Patent no. 7,041,635; or col. 4, lines 2553, of U.S. Patent No. 6,458,563. In some embodiments, a B-domain-deleted FVIII has a deletion of most of the B domain, but still contains amino-terminal

-992018203206 08 May 2018 [0179] sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91/09122. In some embodiments, a B-domain-deleted FVIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R.C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990). A Bdomain-deleted Factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of FVIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988). Additional B domain deletions that are part of the invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 through 1648 (Pasek (PCT application No.88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:1625, EP 295597)). In other embodiments, BDD FVIII includes a FVIII polypeptide containing fragments of the B-domain that retain one or more N-linked glycosylation sites, e.g., residues 757, 784, 828, 900, 963, or optionally 943, which correspond to the amino acid sequence of the full-length FVIII sequence. Examples of the B-domain fragments include 226 amino acids or 163 amino acids of the B-domain as disclosed in Miao, H.Z., et al., Blood 103(a): 3412-3419 (2004), Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359 (2008), and Pipe, S.W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011) (i.e., the first 226 amino acids or 163 amino acids of the B domain are retained). In still other embodiments, BDD FVIII further comprises a point mutation at residue 309 (from Phe to Ser) to improve expression of the BDD FVIII protein. See Miao, H.Z., et al., Blood 103(a): 3412-3419 (2004). In still other embodiments, the BDD FVIII includes a FVIII polypeptide containing a portion of the B-domain, but not containing one or more furin cleavage sites (e.g., Argl313 and Arg 1648). See Pipe, S.W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011). Each of the foregoing deletions may be made in any FVIII sequence.

In some embodiments, the FVIII has a partial B-domain. In some embodiments, the FVIII protein with a partial B-domain is FVIII 198 (SEQ ID NO: 89). FVIII198 is a partial B-domain containing single chain FVIIIFc

- 1002018203206 08 May 2018 [0180] [0181] [0182] [0183] molecule-226N6. 226 represents the N-terminus 226 amino acid of the FVIII Bdomain, and N6 represents six N-glycosylation sites in the B-domain.

In one embodiment, FVIII is cleaved right after arginine at amino acid 1648 (in full-length Factor VIII or SEQ ID NO: 4), amino acid 754 (in the S743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 6), or the corresponding arginine residue (in other variants), thereby resulting in a heavy chain and a light chain. In another embodiment, FVIII comprises a heavy chain and a light chain, which are linked or associated by a metal ion-mediated noncovalent bond.

In other embodiments, FVIII is a single chain FVIII that has not been cleaved right after Arginine at amino acid 1648 (in full-length FVIII or SEQ ID NO: 4), amino acid 754 (in the S743/Q1638 B-domain-deleted FVIII or SEQ ID NO: 6), or the corresponding Arginine residue (in other variants). A single chain FVIII may comprise one or more amino acid substitutions. In one embodiment, the amino acid substitution is at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6). The amino acid substitution can be any amino acids other than arginine, e.g., isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine.

FVIII can further be cleaved by thrombin and then activated as FVIIIa, serving as a cofactor for activated Factor IX (FIXa). And the activated FIX together with activated FVIII forms a Xase complex and converts Factor X to activated Factor X (FXa). For activation, FVIII is cleaved by thrombin after three Arginine residues, at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deleted FVIII sequence), the cleavage generating FVIIIa having the 50kDa Al, 43kDa A2, and 73kDa A3-C1-C2 chains In one embodiment, the FVIII protein useful for the present invention is nonactive FVIII. In another embodiment, the FVIII protein is an activated FVIII.

The protein having FVIII polypeptide linked to or associated with the VWF fragment can comprise a sequence at least 50%, 60%, 70%, 80%, 90%,

- 101 2018203206 08 May 2018 [0184] [0185]

95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or 6, wherein the sequence has the FVIII clotting activity, e.g., activating Factor IX as a cofactor to convert Factor X to activated Factor X (FXa).

Hybrid or chimeric polypeptides and proteins, as used herein, includes a combination of a first polypeptide chain, e.g., the VWF fragment, optionally fused to a first Ig constant region or a portion thereof, with a second polypeptide chain, e.g., a FVIII protein linked to an XTEN sequence, optionally fused to a second Ig constant region or a portion thereof, thereby forming a heterodimer. In one embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via protein-protein interactions, such as charge-charge or hydrophobic interactions. In another embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via disulfide or other covalent bond(s). Hybrids are described, for example, in US 2004/101740 and US 2006/074199. The second polypeptide may be an identical copy of the first polypeptide or a non-identical polypeptide. In one embodiment, the first polypeptide is a FVIII protein(X)-Fc fusion protein, and the second polypeptide is a polypeptide comprising, consisting essentially of, or consisting of an Fc region, wherein the first polypeptide and the second polypeptide are associated with each other. In another embodiment, the first polypeptide comprises a VWF ffagmentXTEN-Fc fusion protein, and the second polypeptide comprises FVIII-Fc fusion protein, making the hybrid a heterodimer. In other embodiments, the first polypeptide comprises a VWF ffagment-Fc fusion protein, and the second polypeptide comprises FVIII(X)-Fc fusion protein, making the hybrid a heterodimer. In yet other embodiments, the first polypeptide comprises a VWF fragment-XTEN-Fc fusion protein, and the second polypeptide comprises FVIII(X)-Fc fusion protein. The first polypeptide and the second polypeptide can be associated through a covalent bond, e.g., a disulfide bond, between the first Fc region and the second Fc region. The first polypeptide and the second polypeptide can further be associated with each other by binding between the VWF fragment and the FVIII protein.

A FVIII protein useful in the present invention can include FVIII having one or more additional XTEN sequences, which do not affect the FVIII coagulation activity. Such XTEN sequences can be fused to the C-terminus or N- 1022018203206 08 May 2018 [0186] [0187] terminus of the FVIII protein or inserted between one or more of the two amino acid residues in the FVIII protein wherein the insertions do not affect the FVIII coagulation activity or FVIII function. In one embodiment, the insertions improve pharmacokinetic properties of the FVIII protein (e.g., half-life). In another embodiment, the insertions can be multiple insertions, e.g., more than two, three, four, five, six, seven, eight, nine, or ten insertions. Examples of the insertion sites include, but are not limited to, the sites listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, 15 or any combinations thereof.

The FVIII protein linked to one or more XTEN sequences can be represented as FVIII(X), FVIII(Xl), FVIII^-X-FVIII^ wherein FVIII^ comprises, consists essentially of, or consists of a first portion of a FVIII protein from amino acid residue a to amino acid residue b; X or XI comprises, consists essentially of, or consists of one or more XTEN sequences, FVIIΙ^-,ψ comprises, consists essentially of, or consists of a second portion of a FVIII protein from amino acid residue c to amino acid residue d;

a is the N-terminal amino acid residue of the first portion of the FVIII protein, b is the C-terminal amino acid residue of the first portion of the FVIII protein but is also the N-terminal amino acid residue of the two amino acids of an insertion site in which the XTEN sequence is inserted, c is the N-terminal amino acid residue of the second portion of the FVIII protein but is also the C-terminal amino acid residue of the two amino acids of an insertion site in which the XTEN sequence is inserted, and d is the C-terminal amino acid residue of the FVIII protein, and wherein the first portion of the FVIII protein and the second portion of the FVIII protein are not identical to each other and are of sufficient length together such that the FVIII protein has a FVIII coagulation activity.

In one embodiment, the first portion of the FVIII protein and the second portion of the FVIII protein are fragments of SEQ ID NO: 4 [full length mature FVIII sequence] or SEQ ID NO: 6 [B-domain deleted FVIII], e.g., N-terminal portion and C-terminal portion, respectively. In certain embodiments, the first portion of the FVIII protein comprises the Al domain and the A2 domain of the FVIII protein. The second portion of the FVIII protein comprises the A3 domain, the Cl domain, and optionally the C2 domain. In yet other embodiments, the first

- 103 2018203206 08 May 2018 [0188] portion of the FVIII protein comprises the Al domain and A2 domain, and the second portion of the FVIII protein comprises a portion of the B domain, the A3 domain, the Cl domain, and optionally the C2 domain. In still other embodiments, the first portion of the FVIII protein comprises the Al domain, A2 domain, and a portion of the B domain of the FVIII protein, and the second portion of the FVIII protein comprises the A3 domain, the Cl domain, and optionally the C2 domain. In still other embodiments, the first portion of the FVIII protein comprises the Al domain, A2 domain, and a first portion of the B domain of the FVIII protein. The second portion of the FVIII protein comprises a second portion of the B domain, the A3 domain, the C1 domain, and optionally the C2 domain. In some embodiments, the two amino acids (b and c) can be any one or more of the amino acid residues insertion sites shown in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15. For example, b can be the amino acid residue immediately upstream of the site in which one or more XTEN sequences are inserted or linked, and c can be the amino acid residue immediately downstream of the site in which the one or more XTEN sequences are inserted or linked. In some embodiments, a is the first mature amino acid sequence of a FVIII protein, and d is the last amino acid sequence of a FVIII protein. For example,

FVIcan be an amino acid sequence at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 745 of SEQ ID NO: 6 [B domain deleted FVIII amino acid sequence] or SEQ ID NO: 4 [full length FVIII] and FVIII^ can be amino acids 746 to 1438 of SEQ ID NO: 6 or amino acids 1641 to 2332 of SEQ ID NO: 4, respectively.

In some aspects, the insertion site in the FVIII protein is located in one or more domains of the FVIII protein, which is the N-terminus, the Al domain, the A2 domain, the A3 domain, the B domain, the Cl domain, the C2 domain, the Cterminus, or two or more combinations thereof or between two domains of the FVIII protein, which are the Al domain and al acidic region, and the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, and the A3 domain and C1 domain, the Cl domain and C2 domain, or any combinations thereof. For example, the insertion sites in which the XTEN sequence can be inserted are selected from the group consisting of the N-terminus and Al domain, the N-terminus and A2

- 1042018203206 08 May 2018 [0189] [0190] [0191] domain, the N-terminus and A3 domain, the N-terminus and B domain, the Nterminus and Cl domain, the N-terminus and C2 domain, the N-terminus and the C-terminus, the Al and A2 domains, the Al and A3 domains, the Al and B domains, the Al and Cl domains, the Al and C2 domains, the Al domain and the C-terminus, the A2 and A3 domains, the A2 and B domains, the A2 and Cl domains, the A2 and C2 domains, the A2 domain and the C-terminus, the A3 and B domains, the A3 and C1 domains, the A3 and C2 domains, the A3 domain and the C-terminus, the B and Cl domains, the B and C2 domains, the B domain and the C-terminus, the Cl and C2 domains, the Cl and the C-terminus, the C2 domain, and the C-terminus, and two or more combinations thereof. Non-limiting examples of the insertion sites are listed in Tables 7, 8, 9, 10, 11, 12, 13, 14, and 15.

The FVIII protein, in which the XTEN sequence is inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein or linked at the C-terminus or the N-terminus, retains the FVIII activity after linkage to or insertion by the XTEN sequence. The XTEN sequence can be inserted in the FVIII protein once or more than once, twice, three times, four times, five times, or six times such that the insertions do not affect the FVIII activity (i.e., the FVIII protein still retains the coagulation property).

The FVIII protein useful in the present invention can be linked to one or more XTEN polypeptides at the N-terminus or C-terminus of the FVIII protein by an optional linker or inserted immediately downstream of one or more amino acids (e.g., one or more XTEN insertion sites) in the FVIII protein by one or more optional linkers. In one embodiment, the two amino acid residues in which the XTEN sequence is inserted or the amino acid residue to which the XTEN sequence is linked correspond to the two or one amino acid residues of SEQ ID NO: 4 [full length mature FVIII] selected from the group consisting of the residues in Table 7, Table 8, Table 9, and Table 10 and any combinations thereof.

In other embodiments, at least one XTEN sequence is inserted in any one or more XTEN insertion sites disclosed herein or any combinations thereof. In one aspect, at least one XTEN sequence is inserted in one or more XTEN insertion sites disclosed in one or more amino acids disclosed in Table 7.

- 105 2018203206 08 May 2018

TABLE 7: Exemplary XTEN Insertion Sites

11111111	llllllllll Insertion Point*	insertion Residue	FVIII BDD Downstream Sequence	FVIII Domain
1	0	(N- terminus)	ATR	Al
2	3	R	RYY	Al
3	17	M	QSD	Al
4	18	Q	SDL	Al
5	22	G	ELP	Al
6	24	L	PVD	Al
7	26	V	DAR	Al
8	28	A	RFP	Al
9	32	P	RVP	Al
10	38	F	PFN	Al
11	40	F	NTS	Al
12	41	N	TSV	Al
13	60	N	IAK	Al
14	61	I	AKP	Al
15	65	R	PPW	Al
16	81	Y	DTV	Al
17	111	G	AEY	Al
18	116	D	QTS	Al
19	119	S	QRE	Al
20	120	Q	REK	Al
21	128	V	FPG	Al
22	129	F	PGG	Al
23	130	P	GGS	Al
24	182	G	SLA	Al
25	185	A	KEK	Al
26	188	K	TQT	Al
27	205	G	KSW	Al
28	210	S	ETK	Al
29	211	E	TKN	Al
30	216	L	MQD	Al
31	220	R	DAA	Al
32	222	A	ASA	Al
33	223	A	SAR	Al
34	224	S	ARA	Al
35	230	K	MHT	Al
36	243	P	GLI	Al
37	244	G	LIG	Al
38	250	R	KSV	Al
39	318	D	GME	Al
40	333	P	QLR	Al
42	334	Q	LRM	Al
43	336	R	MKN	al

- 1062018203206 08 May 2018

11111111	llllllllll Insertion Point*	Insertion Residue	FVIII BDD Downstream Sequence	FVIII Domain
44	339	N	NEE	al
45	345	D	YDD	al
46	357	V	VRF	al
47	367	s	FIQ	al
48	370	s	RPY	al
49	375	A	KKH	A2
50	376	K	KHP	A2
51	378	H	PKT	A2
52	399	V	LAP	A2
53	403	D	DRS	A2
54	405	R	SYK	A2
55	409	S	QYL	A2
56	416	P	QRI	A2
57	434	E	TFK	A2
58	438	T	REA	A2
59	441	A	IQH	A2
60	442	I	QHE	A2
61	463	I	IFK	A2
62	487	Y	SRR	A2
63	490	R	LPK	A2
64	492	P	KGV	A2
65	493	K	GVK	A2
66	494	G	VKH	A2
67	500	D	FPI	A2
68	506	G	EIF	A2
69	518	E	DGP	A2
70	556	K	ESV	A2
71	565	Q	IMS	A2
72	566	I	MSD	A2
73	598	P	AGV	A2
74	599	A	GVQ	A2
75	603	L	EDP	A2
76	616	s	ING	A2
77	686	G	LWI	A2
78	713	K	NTG	A2
79	719	Y	EDS	A2
80	730	L	LSK	A2
81	733	K	NNA	A2
82	745	N	PPV**	B
83	1640	P	PVL	B
84	1652	R	TTL	B
85	1656	Q	SDQ	A3
86	1685	N	QSP	A3
87	1711	M	sss	A3
88	1713	S	SPH	A3

- 1072018203206 08 May 2018

11111111	IllBlllll! Insertion Point*	Insertion Residue	FVIII BDD Downstream Sequence	FVIII Domain
89	1720	N	RAQ	A3
90	1724	S	GSV	A3
91	1725	G	SVP	A3
92	1726	S	VPQ	A3
93	1741	G	SFT	A3
94	1744	T	QPL	A3
95	1749	R	GEL	A3
96	1773	V	TFR	A3
97	1792	Y	EED	A3
98	1793	E	EDQ	A3
99	1796	Q	RQG	A3
100	1798	Q	GAE	A3
101	1799	G	AEP	A3
102	1802	P	RKN	A3
103	1803	R	KNF	A3
104	1807	V	KPN	A3
105	1808	K	PNE	A3
106	1827	K	DEF	A3
107	1844	E	KDV	A3
108	1861	N	TLN	A3
109	1863	L	NPA	A3
110	1896	E	RNC	A3
111	1900	R	APC	A3
112	1904	N	IQM	A3
113	1905	I	QME	A3
114	1910	P	TFK	A3
115	1920	A	ING	A3
116	1937	D	QRI	A3
117	1981	G	VFE	A3
118	2019	N	KCQ	A3
119	2020	K	CQT	Cl
120	2044	G	QWA	Cl
121	2068	F	SWI	Cl
122	2073	V	DLL	Cl
123	2090	R	QKF	Cl
124	2092	K	FSS	Cl
125	2093	F	SSL	Cl
126	2111	K	WQT	Cl
127	2115	Y	RGN	Cl
128	2120	T	GTL	Cl
129	2125	V	FFG	Cl
130	2171	L	NSC	Cl
131	2173	s	CSM	C2
132	2188	A	QIT	C2
133	2223	V	NNP	C2

- 108 2018203206 08 May 2018

11111111	llllllllll Insertion Point*	Insertion Residue	FVIII BDD Downstream Sequence	FVIII Domain
134	2224	N	NPK	C2
135	2227	K	EWL	C2
136	2268	G	HQW	C2
137	2277	N	GKV	C2
138	2278	G	KVK	C2
139	2290	F	TPV	C2
140	2332	Y	C terminus of FVIII	CT

* Indicates an insertion point for XTEN based on the amino acid number of mature fulllength human FVIII, wherein the insertion could be either on the N- or C-terminal side of the indicated amino acid.

[0192] In some embodiments, one or more XTEN sequences are inserted within about six amino acids up or down from amino acids 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905, or 1910, corresponding to SEQ ID NO: 4 or any combinations thereof.

TABLE 8. Exemplary XTEN Insertion Ranges

IIHIIII	llllllllll Insertion Point	Insertion Residue	FVIII BDD Downstrea m Sequence	FVIII Domain	Distance from insertion residue*
9	32	P	RVP	Al	-3,+6
31	220	R	DAA	Al	-
34	224	S	ARA	Al	+5
43	336	R	MKN	al	-1,+6
44	339	N	NEE	al	-4, +5
52	399	V	LAP	A2	-6, +3
56	416	P	QRI	A2	+6
75	603	L	EDP	A2	6, +6
85	1656	Q	SDQ	B	-3,+6
87	1711	M	sss	A3	-6,+1
91	1725	G	SVP	A3	+6
113	1905	I	QME	A3	+6
114	1910	P	TFK	A3	-5,+6

*Distance from insertion residue refers to the relative number of amino acids away from the N-terminus (negative numbers) or C-terminus (positive numbers) of the designated insertion residue (residue 0) where an insertion may be made. The designation -x refers to an insertion site which is x amino acids away on the Nterminal side of the designated insertion residue. Similarly, the designation +x

- 109refers to an insertion site which is x amino acids away on the C-terminal side of the designated insertion residue.

For example, -1, +2 indicates that the insertion is made at the N-terminus or Cterminus of amino acid residues denoted -1, 0, +1 or +2.

[0193] In other embodiments, one or more XTEN sequences are inserted immediately down stream of one or more amino acids corresponding to the fulllength mature human FVIII selected from the group consisting of one or more insertion sites in Table 9.

TABLE 9. Exemplary XTEN Insertion Sites or Ranges

2018203206 08 May 2018

iiiiiiii	XTEN Insertion Point Range*	First Insertion Residue	IYIII Domain
3	18-32	Q	Al
8	40	F	Al
18	211-224	E	Al
27	336-403	R	Al, A2
43	599	A	A2
47	745-1640	N	B
50	1656-1728	Q	B, a3, A3
57	1796-1804	R	A3
65	1900-1912	R	A3
81	2171-2332	L	C1,C2

* indicates range of insertion sites numbered relative to the amino acid number of mature human FVIII [0194] In yet other embodiments, one or more XTENs are inserted in the B domain of FVIII. In one example, an XTEN is inserted between amino acids 740 and 1640 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 740 and 1640 is optionally not present. In another example, an XTEN is inserted between amino acids 741 and 1690 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 740 and 1690 is optionally not present. In other examples, an XTEN is inserted between amino acids 741 and 1648 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 741 and 1648 is optionally not present. In yet other examples, an XTEN is inserted between amino acids 743 and 1638 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 743 and 1638 is optionally not present. In still other examples, an XTEN is inserted between

- 1102018203206 08 May 2018 [0195] amino acids 745 and 1656 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1656 is optionally not present. In some examples, an XTEN is inserted between amino acids 745 and 1657 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1657 is optionally not present. In certain examples, an XTEN is inserted between amino acids 745 and 1667 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1667 is optionally not present. In still other examples, an XTEN is inserted between amino acids 745 and 1686 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 745 and 1686 is optionally not present. In some other examples, an XTEN is inserted between amino acids 747 and 1642 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 747 and 1642 is optionally not present. In still other examples, an XTEN is inserted between amino acids 751 and 1667 corresponding to SEQ ID NO: 4, wherein the FVIII sequence between amino acids 751 and 1667 is optionally not present.

In some embodiments, one or more XTENs are inserted in one or more amino acids immediately downstream of an amino acid of an insertion site selected from the group consisting of the amino acid residues in Table 10.

- Ill TABLE 10: FVIII XTEN insertion sites and construct designations

2018203206 08 May 2018

Construct Number	Domain	Upstream Residue lllOdlllill	Downstream Residue iiiiililiiii	Upstream Sequence	Downstream Sequence
F8X-1	Al	3	4	ATR	RYY
F8X-2	Al	18	19	YMQ	SDL
F8X-3	Al	22	23	DLG	ELP
F8X-4	Al	26	27	LPV	DAR
F8X-5	Al	40	41	FPF	NTS
F8X-6	Al	60	61	LFN	IAK
F8X-7	Al	116	117	YDD	QTS
F8X-8	Al	130	131	VFP	GGS
F8X-9	Al	188	189	KEK	TQT
F8X-10	Al	216	217	NSL	MQD
F8X-11	Al	230	231	WPK	MHT
F8X-12	Al	333	334	EEP	QLR
F8X-13	A2	375	376	SVA	KKH
F8X-14	A2	403	404	APD	DRS
F8X-15	A2	442	443	EAI	QHE
F8X-16	A2	490	491	RRL	PKG
F8X-17	A2	518	519	TVE	DGP
F8X-18	A2	599	600	NPA	GVQ
F8X-19	A2	713	714	CDK	NTG
F8X-20	BD	745	746	SQN	PPV
F8X-21	BD	745	746	SQN	PPV
F8X-22	BD**	745	746	SQN	PPV
F8X-23	A3	1720	1721	APT	KDE
F8X-24	A3	1796	1797	EDQ	RQG
F8X-25	A3	1802	1803	AEP	RKN
F8X-26	A3	1827	1828	PTK	DEF
F8X-27	A3	1861	1862	HTN	TLN
F8X-28	A3	1896	1897	NME	RNC
F8X-29	A3	1900	1901	NCR	APC
F8X-30	A3	1904	1905	PCN	IQM
F8X-31	A3	1937	1938	AQD	QRI
F8X-32	Cl	2019	2020	YSN	KCQ
F8X-33	Cl	2068	2069	EPF	SWI
F8X-34	Cl	2111	2112	GKK	WQT
F8X-35	Cl	2120	2121	NST	GTL
F8X-36	C2	2171	2172	CDL	NSC
F8X-37	C2	2188	2189	SDA	QIT
F8X-38	C2	2227	2228	NPK	EWL
F8X-39	C2	2277	2278	FQN	GKV
F8X-40	CT	2332	NA	DLY	NA
F8X-41	CT	2332	NA	DLY	NA
F8X-42	Al	3	4	ATR	ATR
pSDOOOl	A2	403	404
pSD0002	A2	599	600

- 1122018203206 08 May 2018

pSD0021	N-term	0	1
pSD0022	Al	32	33
pSD0023	Al	65	66
pSD0024	Al	81	82
pSD0025	Al	119	120
pSD0026	Al	211	212
pSD0027	Al	220	221
pSD0028	Al	224	225
pSD0029	Al	336	337
pSD0030	Al	339	340
pSD0031	A2	378	379
pSD0032	A2	399	400
pSD0033	A2	409	410
pSD0034	A2	416	417
pSD0035	A2	487	488
pSD0036	A2	494	495
pSD0037	A2	500	501
pSD0038	A2	603	604
pSD0039	A3	1656	1657
pSD0040	A3	1711	1712
pSD0041	A3	1725	1726
pSD0042	A3	1749	1750
pSD0043	A3	1905	1906
pSD0044	A3	1910	1911
pDS0062	A3	1900	1901

* Indicates the amino acid number of the mature FVIII protein [0196]

In one embodiment, the one or more XTEN insertion sites are located within one or more surface-exposed, flexible loop structure of the FVIII protein (e.g., a permissive loop). For example, at least one XTEN sequence can be inserted in each FVIII A domain comprising at least two permissive loops into which at least one XTEN polypeptide can be inserted without eliminating procoagulant activity of the recombinant protein, or the ability of the recombinant proteins to be expressed in vivo or in vitro in a host cell. The permissive loops are regions that allow insertion of at least one XTEN sequence with, among other attributes, high surface or solvent exposure and high conformational flexibility. The Al domain comprises a permissive loop-1 (Al-1) region and a permissive loop-2 (A 1-2) region, the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region, the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region.

- 113 2018203206 08 May 2018 [0197] [0198]

In one aspect, a first permissive loop in the FVIII Al domain (Al-1) is located between beta strand 1 and beta strand 2, and a second permissive loop in the FVIII A2 domain (Al-2) is located between beta strand 11 and beta strand 12. A first permissive loop in the FVIII A2 domain (A2-1) is located between beta strand 22 and beta strand 23, and a second permissive loop in the FVIII A2 domain (A2-2) is located between beta strand 32 and beta strand 33. A first permissive loop in the FVIII A3 domain (A3-1) is located between beta strand 38 and beta strand 39, and a second permissive loop in the FVIII A3 (A3-2) is located between beta strand 45 and beta strand 46. In certain aspects, the surface-exposed, flexible loop structure comprising Al-1 corresponds to a region in native mature human FVIII from about amino acid 15 to about amino acid 45 of SEQ IDNO: 4, e.g., from about amino acid 18 to about amino acid 41 of SEQ ID NO: 4. In other aspects, the surface-exposed, flexible loop structure comprising Al-2 corresponds to a region in native mature human FVIII from about amino acid 201 to about amino acid 232 of SEQ ID NO: 4, e.g., from about amino acid 218 to about amino acid 229 of SEQ ID NO: 4. In yet other aspects, the surface-exposed, flexible loop structure comprising A2-1 corresponds to a region in native mature human FVIII from about amino acid 395 to about amino acid 421 of SEQ ID NO: 4, e.g. from about amino acid 397 to about amino acid 418 of SEQ ID NO: 4. In still other embodiments, the surface-exposed, flexible loop structure comprising A2-2 corresponds to a region in native mature human FVIII from about amino acid 577 to about amino acid 635 of SEQ ID NO: 4, e.g., from about amino acid 595 to about amino acid 607 of SEQ ID NO: 4. In certain aspects the surface-exposed, flexible loop structure comprising A3-1 corresponds to a region in native mature human FVIII from about amino acid 1705 to about amino acid 1732 of SEQ ID NO: 4, e.g., from about amino acid 1711 to about amino acid 1725 of SEQ ID NO: 4. In yet other aspects, the surface-exposed, flexible loop structure comprising A3-2 corresponds to a region in native mature human FVIII from about amino acid 1884 to about amino acid 1917 of SEQ ID NO: 4, e.g., from about amino acid 1899 to about amino acid 1911 of SEQ ID NO: 4.

In another embodiment, the one or more amino acids in which at least one XTEN sequence is inserted is located within a3 domain, e.g., amino acids 1649 to 1689, corresponding to full-length mature FVIII polypeptide. In a particular

- 114embodiment, an XTEN sequence is inserted between amino acids 1656 and 1657 of SEQ ID NO: 4 (full-length mature FVIII). In a specific embodiment, a FVIII protein comprising an XTEN sequence inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 further comprises a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

In some embodiments, the one or more insertion sites for one or more XTEN insertions are immediately downstream of one or more amino acids selected from the group consisting of:

2018203206 08 May 2018 [0199]

(1) amino acid 3,	(2) amino acid 18,	(3) amino acid 22,
(4) amino acid 26,	(5) amino acid 32,	(6) amino acid 40,
(7) amino acid 60,	(8) amino acid 65,	(9) amino acid 81,
(10) amino acid 116,	(11) amino acid 119,	(12) amino acid 130,
(13) amino acid 188,	(14) amino acid 211,	(15) amino acid 216,
(16) amino acid 220,	(17) amino acid 224,	(18) amino acid 230,
(19) amino acid 333,	(20) amino acid 336,	(21) amino acid 339,
(22) amino acid 375,	(23) amino acid 399,	(24) amino acid 403,
(25) amino acid 409,	(26) amino acid 416,	(26) amino acid 442,
(28) amino acid 487,	(29) amino acid 490,	(30) amino acid 494,
(31) amino acid 500,	(32) amino acid 518,	(33) amino acid 599,
(34) amino acid 603,	(35) amino acid 713,	(36) amino acid 745,
(37) amino acid 1656, amino acid 1720,	(38) amino acid 1711,	(39)
(40) amino acid 1725, amino acid 1796,	(41) amino acid 1749,	(42)
(43) amino acid 1802, amino acid 1861,	(44) amino acid 1827,	(45)
(46) amino acid 1896, amino acid 1904,	(47) amino acid 1900,	(48)
(49) amino acid 1905, amino acid 1937,	(50) amino acid 1910,	(51)
(52) amino acid 2019, amino acid 2111,	(53) amino acid 2068,	(54)

- 115 2018203206 08 May 2018 [0200] (57) (55) amino acid 2120, (56) amino acid 2171, amino acid 2188, (58) amino acid 2227, (59) amino acid 2277, and (60) two or more combinations thereof.

In one embodiment, a FVIII protein useful for the invention comprises two

XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site and a second XTEN inserted into a second XTEN insertion site. Non-limiting examples of the first XTEN insertion site and the second XTEN insertion site are listed in Table 11.

TABLE 11. Exemplary Insertion Sites for Two XTENs

Insertion I	Insertion 2
Insertion Site	Domain	Insertion Site	Domain
745	B	2332	CT
26	Al	403	A2
40	Al	403	A2
18	Al	403	A2
26	Al	599	A2
40	Al	599	A2
18	Al	599	A2
1720	A3	1900	A3
1725	A3	1900	A3
1711	A3	1905	A3
1720	A3	1905	A3
1725	A3	1905	A3
1656	A3	26	Al
1656	A3	18	Al
1656	A3	40	Al
1656	A3	399	A2
1656	A3	403	A2
1656	A3	1725	A3
1656	A3	1720	A3
1900	A3	18	Al
1900	A3	26	Al
1900	A3	40	Al
1905	A3	18	Al
1905	A3	40	Al
1905	A3	26	Al
1910	A3	26	Al
18	Al	399	A2
26	Al	399	A2
40	Al	399	A2
18	Al	403	A2

- 1162018203206 08 May 2018

Insertion 1	Insertion 2
Insertion Site	Domain	Insertion Site	Domain
1656	A3	1900	A3
1656	A3	1905	A3
1711	A3	40	Al
1711	A3	26	Al
1720	A3	26	Al
1720	A3	40	Al
1720	A3	18	Al
1725	A3	26	Al
1725	A3	40	Al
1725	A3	18	Al
1720	A3	403	A2
1720	A3	399	A2
1711	A3	403	A2
1720	A3	403	A2
1725	A3	403	A2
1725	A3	399	A2
1711	A3	403	A2
1900	A3	399	A2
1900	A3	403	A2
1905	A3	403	A2
1905	A3	399	A2
1910	A3	403	A2

[0201]

The two XTENs inserted or linked to the FVIII protein can be identical or different. In some embodiments, a FVIII protein useful for the invention comprises two XTEN sequences inserted in the FVIII protein, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4 (the C-terminus). In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 1656, or 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 599 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, 40, 399,

- 1172018203206 08 May 2018 [0202]

403, 1725, 1720, 1900, 1905, or 2332 corresponding to SEQ ID NO: 4. In certain embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1900 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4. In some embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 399 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18, 26, or 40 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a second XTEN sequence inserted immediately downstream of amino acid 18 corresponding to SEQ ID NO: 4. In a particular embodiment, the FVIII protein comprising two XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 745 corresponding to SEQ ID NO: 4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 corresponding to SEQ ID NO: 4, wherein the FVIII protein further has a deletion from amino acid 745 corresponding to SEQ ID NO: 4 to amino acid 1685 corresponding to SEQ ID NO: 4, a mutation or substitution at amino acid 1680 corresponding to SEQ ID NO: 4, e.g., Y1680F, a mutation or substitution at amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, or at least two mutations or substitutions at amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A, and amino acid 1680 corresponding to SEQ ID NO: 4, e.g., Y1680F. In a specific embodiment, the FVIII protein comprises two XTEN sequences, a first XTEN inserted immediately downstream of amino acid 1656 corresponding to SEQ ID NO: 4 and a second XTEN sequence inserted immediately downstream of amino acid 2332 of SEQ ID NO: 4, wherein the FVIII protein further has a deletion from amino acid 745 to amino acid 1656 corresponding to SEQ ID NO: 4.

In certain embodiments, a FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN sequence, and a third XTEN sequence

- 118 2018203206 08 May 2018 inserted into a third XTEN insertion site. The first, second, or third XTEN sequences can be identical or different. The first, second, and third insertion sites can be selected from the group of any one of the insertion sites disclosed herein. In some embodiments, the FVIII protein comprising three XTEN sequences can further comprise a mutation or substitution, e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g., R1648A. For example, non-limiting examples of the first, second, and third XTEN insertion sites are listed in Table 12.

TABLE 12. Exemplary Insertion Sites for Three XTENs

Insertion 1	Insertion 2	Insertion 3
Insertion Site	Domain	Insertion llll/lBlllill	Domain	Insertion lllllliilllll	Domain
26	Al	403	A2	1656	A3
26	Al	403	A2	1720	A3
26	Al	403	A2	1900	A3
26	Al	1656	A3	1720	A3
26	Al	1656	A3	1900	A3
26	Al	1720	A3	1900	A3
403	A2	1656	A3	1720	A3
403	A2	1656	A3	1900	A3
403	A2	1720	A3	1900	A3
1656	A3	1720	A3	1900	A3
745	B	1900		2332	CT
18	Al	745	B	2332	CT
26	Al	745	B	2332	CT
40	Al	745	B	2332	CT
18	Al	745	B	2332	CT
40	Al	745	B	2332	CT
403	A2	745	B	2332	CT
399	A2	745	B	2332	CT
1725	A3	745	B	2332	CT
1720	A3	745	B	2332	CT
1711	A3	745	B	2332	CT
1900	A3	745	B	2332	CT
1905	A3	745	B	2332	CT
1910	A3	745	B	2332	CT

[0203] In some embodiments, a FVIII protein comprises three XTEN sequences, a first XTEN sequence inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence inserted downstream of amino acid 403 corresponding to SEQ ID NO: 4, and a third XTEN sequence inserted downstream of amino acid 1656, 1720, or 1900 corresponding to SEQ ID

- 1192018203206 08 May 2018 [0204]

NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 4. In yet other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 26 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In still other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1656 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1720 or 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 403 or 1656 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 1720 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 1900 corresponding to SEQ ID NO: 4. In other embodiments, the first XTEN sequence is inserted immediately downstream of amino acid 18, 26, 40, 399, 403, 1711, 1720, 1725, 1900, 1905, or 1910 corresponding to SEQ ID NO: 4, a second XTEN sequence is inserted downstream of amino acid 745 corresponding to SEQ ID NO: 4, and a third XTEN sequence is inserted downstream of amino acid 2332 corresponding to SEQ ID NO: 4.

In other embodiments, a FVIII protein in the invention comprises four XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence inserted into a second insertion site, a third XTEN sequence inserted into a third insertion site, and a fourth XTEN sequence inserted into a fourth insertion site. The first, second, third, and fourth XTEN sequences can be identical, different, or combinations thereof. In some embodiments, the FVIII protein comprising four XTEN sequences can further comprise a mutation or substitution, e.g., amino acid 1648 corresponding to SEQ ID NO: 4, e.g.,

- 120R1648A. Non-limiting examples of the first, second, third, and fourth XTEN insertion sites are listed in Table 13.

2018203206 08 May 2018

TABLE 13. Exemplary Insertion Sites for Four XTENs

Insertion 1	Insertion 2	Insertion 3	Insertion 4
Insertion Site	Domain	Insertion llllItBllil	Domai IllOllI!	Insertion llllliBlIll	Domai lllfilll	Insertion llllOlll	Domai illOlll
26	Al	403	A2	1656	a3	1720	A3
26	Al	403	A2	1656	a3	1900	A3
26	Al	403	A2	1720	A3	1900	A3
26	Al	1656	a3	1720	A3	1900	A3
403	A2	1656	a3	1720	A3	1900	A3
0040	Al	0403	A2	745	B	2332	CT
0040	Al	0403	A2	745	B	2332	CT
0018	Al	0409	A2	745	B	2332	CT
0040	Al	0409	A2	745	B	2332	CT
0040	Al	0409	A2	745	B	2332	CT
0018	Al	0409	A2	745	B	2332	CT
0040	Al	1720	A3	745	B	2332	CT
0026	Al	1720	A3	745	B	2332	CT
0018	Al	1720	A3	745	B	2332	CT
0018	Al	1720	A3	745	B	2332	CT
0018	Al	1720	A3	745	B	2332	CT
0026	Al	1720	A3	745	B	2332	CT
0018	Al	1720	A3	745	B	2332	CT
0018	Al	1900	A3	745	B	2332	CT
0018	Al	1900	A3	745	B	2332	CT
0026	Al	1900	A3	745	B	2332	CT
0040	Al	1900	A3	745	B	2332	CT
0040	Al	1905	A3	745	B	2332	CT
0018	Al	1905	A3	745	B	2332	CT
0040	Al	1905	A3	745	B	2332	CT
0026	Al	1905	A3	745	B	2332	CT
0018	Al	1905	A3	745	B	2332	CT
0018	Al	1905	A3	745	B	2332	CT
0018	Al	1910	A3	745	B	2332	CT
0018	Al	1910	A3	745	B	2332	CT
0040	Al	1910	A3	745	B	2332	CT
0026	Al	1910	A3	745	B	2332	CT
0018	Al	1910	A3	745	B	2332	CT
0026	Al	1910	A3	745	B	2332	CT
0040	Al	1910	A3	745	B	2332	CT
0018	Al	1910	A3	745	B	2332	CT
0409	A2	1720	A3	745	B	2332	CT
0403	A2	1720	A3	745	B	2332	CT
0409	A2	1720	A3	745	B	2332	CT
0403	A2	1720	A3	745	B	2332	CT

- 121 2018203206 08 May 2018

0403	A2	1720	A3	745	B	2332	CT
0403	A2	1900	A3	745	B	2332	CT
0403	A2	1900	A3	745	B	2332	CT
0409	A2	1900	A3	745	B	2332	CT
0403	A2	1900	A3	745	B	2332	CT
0403	A2	1900	A3	745	B	2332	CT
0409	A2	1900	A3	745	B	2332	CT
0409	A2	1905	A3	745	B	2332	CT
0403	A2	1905	A3	745	B	2332	CT
0403	A2	1905	A3	745	B	2332	CT
0403	A2	1905	A3	745	B	2332	CT
0409	A2	1905	A3	745	B	2332	CT
0403	A2	1905	A3	745	B	2332	CT
0409	A2	1910	A3	745	B	2332	CT
0403	A2	1910	A3	745	B	2332	CT
0403	A2	1910	A3	745	B	2332	CT
0403	A2	1910	A3	745	B	2332	CT
0403	A2	1910	A3	745	B	2332	CT
1720	A3	1900	A3	745	B	2332	CT
1720	A3	1905	A3	745	B	2332	CT
1720	A3	1910	A3	745	B	2332	CT
1720	A3	1910	A3	745	B	2332	CT
0403	A2	1656	a3	1720	A3	2332	CT
0403	A2	1656	a3	1900	A3	2332	CT
0403	A2	1720	A3	1900	A3	2332	CT
1656	a3	1720	A3	1900	A3	2332	CT
0018	Al	0403	A2	1656	a3	2332	CT
0018	Al	0403	A2	1720	A3	2332	CT
0018	Al	0403	A2	1900	A3	2332	CT
0018	Al	1656	a3	1720	A3	2332	CT
0018	Al	1656	a3	1900	A3	2332	CT
0018	Al	1720	A3	1900	A3	2332	CT
0018	Al	0403	A2	0745	B	2332	CT
0018	Al	0745	B	1720	A3	2332	CT
0018	Al	0745	B	1900	A3	2332	CT
0403	A2	0745	B	1720	A3	2332	CT
0403	A2	0745	B	1900	A3	2332	CT
0745	B	1720	A3	1900	A3	2332	CT
0188	Al	1900	A3	0745	B	2332	CT
0599		1900	A3	0745	B	2332	CT
2068		1900	A3	0745	B	2332	CT
2171		1900	A3	0745	B	2332	CT
2227		1900	A3	0745	B	2332	CT
2277		1900	A3	0745	B	2332	CT

[0205] In some embodiments, a FVIII protein comprises five XTEN sequences, a first XTEN sequence inserted into a first insertion site, a second XTEN sequence

- 122inserted into a second insertion site, a third XTEN sequence inserted into a third

XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, and a fifth XTEN sequence inserted into a fifth XTEN insertion site.

The first, second, third, fourth, of fifth XTEN sequences can be identical, different, or combinations thereof Non-limiting examples of the first, second,

2018203206 08 May 2018 third, fourth, and fifth insertion sites are listed in Table 14.

TABLE 14. Exemplary Insertion Sites for Five XTENs

XTEN Insertion 1	XTEN insertion	XTEN Insertion 3	XTEN Insertion 4	XTEN Insertion 5
0403	1656	1720	1900	2332
0018	0403	1656	1720	2332
0018	0403	1656	1900	2332
0018	0403	1720	1900	2332
0018	1656	1720	1900	2332
0018	0403	0745	1720	2332
0018	0403	0745	1900	2332
0018	0745	1720	1900	2332
0403	0745	1720	1900	2332

[0206] In certain embodiments, a FVIII protein comprises six XTEN sequences, a first XTEN sequence inserted into a first XTEN insertion site, a second XTEN sequence inserted into a second XTEN insertion site, a third XTEN sequence inserted into a third XTEN insertion site, a fourth XTEN sequence inserted into a fourth XTEN insertion site, a fifth XTEN sequence inserted into a fifth XTEN insertion site, and a sixth XTEN sequence inserted into a sixth XTEN insertion site. The first, second, third, fourth, fifth, or sixth XTEN sequences can be identical, different, or combinations thereof Examples of the six XTEN insertion sites include, but are not limited to the insertion sites listed in Table 15.

TABLE 15. Exemplary XTEN Insertion Sites for Six XTENs

XTEN Insertion 1	XTEN insertion 2	XTEN Insertion 3	XTEN Insertion 4	XTEN Insertion 5	XTEN Insertion 6
0018	0403	1656	1720	1900	2332
0018	0403	0745	1720	1900	2332

[0207] In a particular example, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between

- 123 2018203206 08 May 2018 [0208] [0209] amino acids 1720 and 1721 corresponding to SEQ ID NO: 4 (full-length mature FVIII). In another example, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In some examples, a first XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In other examples, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In yet other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In still other embodiments, a first XTEN is inserted between amino acids 403 and 404 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4. In certain embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4, and a third XTEN is inserted between amino acids 1900 and 1901 corresponding to SEQ ID NO: 4. In some embodiments, a first XTEN is inserted between amino acids 26 and 27 corresponding to SEQ ID NO: 4, a second XTEN is inserted between amino acids 1656 and 1657 corresponding to SEQ ID NO: 4, a third XTEN is inserted between amino acids 1720 and 1721 corresponding to SEQ ID NO: 4, and a fourth XTEN is inserted between 1900 and 1901 corresponding to SEQ ID NO: 4.

In a particular embodiment, an XTEN sequence is inserted between amino acids 745 and 746 of a full-length Factor VIII or the corresponding insertion site of the B-domain deleted Factor VIII.

In some embodiments, a chimeric protein of the invention comprises two polypeptide sequences, a first polypeptide sequence comprising an amino acid

- 1242018203206 08 May 2018 [0210] [0211] [0212] sequence at least about 80%, 90%, 95%, or 100% identical to a sequence selected from FVIII-161 (SEQ ID NO: 101), FVIII-169 (SEQ ID NO: 103), FVIII-170 (SEQ ID NO: 102), FVIII-173 (SEQ ID NO: 104); FVIII-195 (SEQ ID NO: 105); FVIII-196 (SEQ ID NO: 106), FVIII-199 (SEQ ID NO: 107), FVIII-201 (SEQ ID NO: 108); FVIII-203 (SEQ ID NO: 109), FVIII-204 (SEQ ID NO: 110), FVIII205 (SEQ ID NO: 111), FVIII-266 (SEQ ID NO: 112), FVIII-267 (SEQ ID NO: 113), FVIII-268 (SEQ ID NO: 114), FVIII-269 (SEQ ID NO: 115), FVIII-271 (SEQ ID NO: 116), or FVIII-272 (SEQ ID NO: 117) and a second polypeptide sequence comprising an amino acid sequence at least about 80%, 90%, 95%, or 100% identical to a sequence selected from VWF031 (SEQ ID NO: 118), VWF034 (SEQ ID NO: 119), or VWF-036 (SEQ ID NO: 120).

D) lg Constant Region or a portion thereof

The VWF fragment or the FVIII protein linked to an XTEN sequence in the present invention can further comprise an lg constant region or a portion thereof. The lg constant region or a portion thereof can improve pharmacokinetic or pharmacodynamic properties of the VWF fragment or the FVIII protein in combination with the XTEN sequence. In certain embodiments, the lg constant region or a portion thereof extends a half-life of a molecule fused to the lg constant region or a portion thereof.

An lg constant region is comprised of domains denoted CH (constant heavy) domains (CHI, CH2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA, IgD, or IgE), the constant region can be comprised of three or four CH domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

An lg constant region or a portion thereof for producing the chimeric protein of the present invention may be obtained from a number of different sources. In some embodiments, an lg constant region or a portion thereof is derived from a human lg. It is understood, however, that the lg constant region or a portion thereof may be derived from an lg of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or nonhuman primate (e.g. chimpanzee, macaque) species. Moreover, the lg constant region or a portion thereof may be derived from any lg class, including IgM, IgG,

- 125 2018203206 08 May 2018 [0213] [0214]

IgD, IgA, and IgE, and any Ig isotype, including IgGl, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgGl is used.

A variety of the Ig constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains sequence can be selected having a particular effector function (or lacking a particular effector function) or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Ig constant region sequences (e.g., hinge, CH2, and/or CH3 sequences, or portions thereof) can be derived from these sequences using art recognized techniques. The genetic material obtained using any of the foregoing methods may then be altered or synthesized to obtain polypeptides of the present invention. It will further be appreciated that the scope of this invention encompasses alleles, variants and mutations of constant region DNA sequences.

The sequences of the Ig constant region or a portion thereof can be cloned, e.g., using the polymerase chain reaction and primers which are selected to amplify the domain of interest. To clone a sequence of the Ig constant region or a portion thereof from an antibody, mRNA can be isolated from hybridoma, spleen, or lymph cells, reverse transcribed into DNA, and antibody genes amplified by PCR. PCR amplification methods are described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g, PCR Protocols: A Guide to Methods and Applications Innis et al. eds., Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol.

217:270). PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes. Numerous primer sets suitable for amplification of antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem.

- 1262018203206 08 May 2018 [0215] [0216] [0217] [0218] [0219]

Biophys. Res. Commun. 160:1250). The cloning of antibody sequences is further described in Newman et al., U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein.

An Ig constant region used herein can include all domains and the hinge region or portions thereof. In one embodiment, the Ig constant region or a portion thereof comprises CH2 domain, CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

As used herein, the term Fc region is defined as the portion of a polypeptide which corresponds to the Fc region of native Ig, i.e., as formed by the dimeric association of the respective Fc domains of its two heavy chains. A native Fc region forms a homodimer with another Fc region. In contrast, the term genetically-fused Fc region or single-chain Fc region (scFc region), as used herein, refers to a synthetic dimeric Fc region comprised of Fc domains genetically linked within a single polypeptide chain (i.e., encoded in a single contiguous genetic sequence).

In one embodiment, the Fc region refers to the portion of a single Ig heavy chain beginning in the hinge region just upstream of the papain cleavage site (i.e. residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

The Fc region of an Ig constant region, depending on the Ig isotype can include the CH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Fc region of an Ig bestow several desirable properties on a chimeric protein including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which are incorporated herein by reference in their entireties.

An Ig constant region or a portion thereof can be an FcRn binding partner. FcRn is active in adult epithelial tissues and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). An FcRn binding partner is a portion of an Ig that binds to FcRn.

- 1272018203206 08 May 2018 [0220] [0221] [0222]

The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, monkey FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn receptor binds IgG (but not other Ig classes such as IgA, IgM, IgD, and IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to serosal direction, and then releases the IgG at relatively higher pH found in the interstitial fluids. It is expressed in adult epithelial tissue (U.S. Pat. Nos.

6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1) including lung and intestinal epithelium (Israel et al. 1997, Immunology 92:69) renal proximal tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358) as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

FcRn binding partners useful in the present invention encompass molecules that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of Igs or Ig fragments, or regions, are all based on Rabat et al. 1991, Sequences of Proteins of Immunological Interest, U.S. Department of Public Health, Bethesda, Md.

Fc regions or FcRn binding partners bound to FcRn can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an Fc region or an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo

- 128 2018203206 08 May 2018 [0223] [0224] [0225] half-life of these proteins. In certain embodiments, the portions of Ig constant regions are an Fc region or an FcRn binding partner that typically associates, via disulfide bonds and other non-specific interactions, with another Fc region or another FcRn binding partner to form dimers and higher order multimers.

Two FcRn receptors can bind a single Fc molecule. Crystallographic data suggest that each FcRn molecule binds a single polypeptide of the Fc homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc fragment of an IgG, to a biologically active molecule provides a means of delivering the biologically active molecule orally, buccally, sublingually, rectally, vaginally, as an aerosol administered nasally or via a pulmonary route, or via an ocular route. In another embodiment, the chimeric protein can be administered invasively, e.g., subcutaneously, intravenously.

An FcRn binding partner region is a molecule or a portion thereof that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the Fc region. Specifically bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 10⁶ M’¹, or higher than 10⁸ M’¹. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

In certain embodiments, a chimeric protein of the invention comprises one or more truncated Fc regions that are nonetheless sufficient to confer Fc receptor (FcR) binding properties to the Fc region. For example, the portion of an Fc region that binds to FcRn (i.e., the FcRn binding portion) comprises from about amino acids 282-438 of IgGl, EU numbering (with the primary contact sites being amino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 ofthe CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. Thus, an Fc region of the invention may comprise or consist of an FcRn binding

- 1292018203206 08 May 2018 [0226] [0227] portion. FcRn binding portions may be derived from heavy chains of any isotype, including IgGl, IgG2, IgG3 and IgG4. In one embodiment, an FcRn binding portion from an antibody of the human isotype IgGl is used. In another embodiment, an FcRn binding portion from an antibody of the human isotype

IgG4 is used.

In another embodiment, the Fc region includes an amino acid sequence of an Fc domain or derived from an Fc domain. In certain embodiments, an Fc region comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain (about amino acids 216-230 of an antibody Fc region according to EU numbering), a CH2 domain (about amino acids 231-340 of an antibody Fc region according to EU numbering), a CH3 domain (about amino acids 341-438 of an antibody Fc region according to EU numbering), a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, an Fc region comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, an Fc region comprises, consists essentially of, or consists of a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a hinge domain (or a portion thereof) fused to a CH2 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof), a CH2 domain (or a portion thereof) fused to both a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In still other embodiments, an Fc region lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). In a particular embodiment, an Fc region comprises or consists of amino acids corresponding to EU numbers 221 to 447.

The Fc regions denoted as F, FI, or F2 herein may be obtained from a number of different sources. In one embodiment, an Fc region of the polypeptide is derived from a human lg. It is understood, however, that an Fc region may be derived from an lg of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, or guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover, the polypeptide of the Fc domains or portions thereof may be derived from any lg class, including IgM, IgG, IgD, IgA and IgE, and any lg isotype, including IgGl, IgG2, IgG3 and IgG4. In another embodiment, the human isotype IgGl is used.

- 1302018203206 08 May 2018 [0228] [0229] [0230]

In certain embodiments, the Fc variant confers a change in at least one effector function imparted by an Fc region comprising said wild-type Fc domain (e.g., an improvement or reduction in the ability of the Fc region to bind to Fc receptors (e.g. FcyRI, FcyRII, or FcyRIII) or complement proteins (e.g. Clq), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variant provides an engineered cysteine residue.

The Fc regions of the invention may employ art-recognized Fc variants which are known to impart a change (e.g., an enhancement or reduction) in effector function and/or FcR or FcRn binding. Specifically, a binding molecule of the invention may include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications W088/07089A1, WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WOOO/32767A1, WO00/42072A2, WO02/44215A2, W002/060919A2, WO03/074569A2, W004/016750A2, W004/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2, WO04/099249A2, W005/040217A2, WO04/044859, W005/070963A1, WO05/077981A2, WO05/092925A2, WO05/123780A2, W006/019447A1, W006/047350A2, and WO06/085967A2; US Patent Publication Nos. US2007/0231329, US2007/0231329, US2007/0237765, US2007/0237766, US2007/0237767, US2007/0243188, US20070248603, US20070286859, US20080057056 ; or US Patents 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091, each of which is incorporated by reference herein. In one embodiment, the specific change (e.g., the specific substitution of one or more amino acids disclosed in the art) may be made at one or more of the disclosed amino acid positions. In another embodiment, a different change at one or more of the disclosed amino acid positions (e.g., the different substitution of one or more amino acid position disclosed in the art) may be made.

The Fc region or FcRn binding partner of IgG can be modified according to well recognized procedures such as site directed mutagenesis and the like to yield modified IgG or Fc fragments or portions thereof that will be bound by

- 131 2018203206 08 May 2018 [0231]

FcRn. Such modifications include modifications remote from the FcRn contact sites as well as modifications within the contact sites that preserve or even enhance binding to the FcRn. For example, the following single amino acid residues in human IgGl Fc (Fc yl) can be substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A,

L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F,

R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where for example P238A represents wild type proline substituted by alanine at position number 238. As an example, a specific embodiment incorporates the N297A mutation, removing a highly conserved N-glycosylation site. In addition to alanine other amino acids may be substituted for the wild type amino acids at the positions specified above. Mutations may be introduced singly into Fc giving rise to more than one hundred Fc regions distinct from the native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together, giving rise to hundreds more Fc regions. Moreover, one of the Fc region of a construct of the invention may be mutated and the other Fc region of the construct not mutated at all, or they both may be mutated but with different mutations.

Certain of the above mutations may confer new functionality upon the Fc region or FcRn binding partner. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing circulating half-life of the Fc region, and to render the Fc region incapable of binding to FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA, without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields

- 1322018203206 08 May 2018 [0232] [0233] [0234] et al. 1995, J. Biol. Chem. 276:6591). As a further example of new functionality arising from mutations described above affinity for FcRn may be increased beyond that of wild type in some instances. This increased affinity may reflect an increased on rate, a decreased off rate or both an increased on rate and a decreased off' rate. Examples of mutations believed to impart an increased affinity for FcRn include, but not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

Additionally, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234237. Therefore, another example of new functionality and potential decreased immunogenicity may arise from mutations of this region, as for example by replacing amino acids 233-236 of human IgGl ELLG to the corresponding sequence from IgG2 PVA (with one amino acid deletion). It has been shown that FcyRI, FcyRII, and FcyRIII, which mediate various effector functions will not bind to IgGl when such mutations have been introduced. Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al. 1999, Eur. J. Immunol. 29:2613.

In one embodiment, the Ig constant region or a portion thereof, e.g, an Fc region, is a polypeptide including the sequence PKNSSMISNTP (SEQ ID NO: 52) and optionally further including a sequence selected from HQSLGTQ (SEQ ID NO: 53), HQNLSDGK (SEQ ID NO: 54), HQNISDGK (SEQ ID NO: 55), or VISSHLGQ (SEQ ID NO: 56) (U.S. Pat. No. 5,739,277).

In another embodiment, the immunoglobulin constant region or a portion thereof comprises an amino acid sequence in the hinge region or a portion thereof that forms one or more disulfide bonds with another immunoglobulin constant region or a portion thereof. The disulfide bond by the immunoglobulin constant region or a portion thereof places the first polypeptide comprising FVIII and the second polypeptide comprising the VWF fragment together so that endogenous VWF does not replace the VWF fragment and does not bind to the FVIII. Therefore, the disulfide bond between the first immunoglobulin constant region or a portion thereof and a second immunoglobulin constant region or a portion thereof prevents interaction between endogenous VWF and the FVIII protein.

This inhibition of interaction between the VWF and the FVIII protein allows the half-life of the FVIII protein to go beyond the two fold limit. The hinge region or

- 133 2018203206 08 May 2018 [0235] [0236] [0237] a portion thereof can further be linked to one or more domains of CHI, CH2,

CH3, a fragment thereof, and any combinations thereof In a particular embodiment, the immunoglobulin constant region or a portion thereof is a hinge region and CH2.

In certain embodiments, the lg constant region or a portion thereof is hemiglycosylated. For example, the chimeric protein comprising two Fc regions or FcRn binding partners may contain a first, glycosylated, Fc region (e.g., a glycosylated CH2 region) or FcRn binding partner and a second, aglycosylated, Fc region (e.g., an aglycosylated CH2 region) or FcRn binding partner. In one embodiment, a linker may be interposed between the glycosylated and aglycosylated Fc regions. In another embodiment, the Fc region or FcRn binding partner is fully glycosylated, i.e., all of the Fc regions are glycosylated. In other embodiments, the Fc region may be aglycosylated, i.e., none of the Fc moieties are glycosylated.

In certain embodiments, a chimeric protein of the invention comprises an amino acid substitution to an lg constant region or a portion thereof (e.g., Fc variants), which alters the antigen-independent effector functions of the lg constant region, in particular the circulating half-life of the protein.

Such proteins exhibit either increased or decreased binding to FcRn when compared to proteins lacking these substitutions and, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered polypeptide is desired, e.g., to treat a chronic disease or disorder (see, e.g., US Patents 7,348,004, 7,404,956, and 7,862,820). In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter halflives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations where the starting polypeptide has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be

- 1342018203206 08 May 2018 [0238] desired include those applications in which localization the brain, kidney, and/or liver is desired. In one exemplary embodiment, the chimeric protein of the invention exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the chimeric protein of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, a protein with altered FcRn binding comprises at least one Fc region or FcRn binding partner (e.g, one or two Fc regions or FcRn binding partners) having one or more amino acid substitutions within the FcRn binding loop of an Ig constant region. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering) of a wild-type, full-length, Fc region. In other embodiments, an Ig constant region or a portion thereof in a chimeric protein of the invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions within the 15 A FcRn contact zone. As used herein, the term 15 A FcRn contact zone includes residues at the following positions of a wild-type, full-length Fc moiety: 243-261, 275-280, 282-293, 302319, 336- 348, 367, 369, 372-389, 391, 393, 408, 424, 425-440 (EU numbering).

In other embodiments, a Ig constant region or a portion thereof of the invention having altered FcRn binding affinity comprises at least one Fc region or FcRn binding partner having one or more amino acid substitutions at an amino acid position corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein.

An Fc region or FcRn binding partner used in the invention may also comprise an art recognized amino acid substitution which alters the glycosylation of the chimeric protein. For example, the Fc region or FcRn binding partner of the chimeric protein linked to a VWF fragment or a FVIII protein may comprise an Fc region having a mutation leading to reduced glycosylation (e.g., N- or O-linked glycosylation) or may comprise an altered glycoform of the wild-type Fc moiety (e.g., a low fucose or fucose-free glycan).

- 135 2018203206 08 May 2018 [0239] [0240] [0241]

In one embodiment, an unprocessed chimeric protein of the invention may comprise a genetically fused Fc region (i.e., scFc region) having two or more of its constituent Ig constant region or a portion thereof independently selected from the Ig constant region or a portion thereof described herein. In one embodiment, the Fc regions of a dimeric Fc region are the same. In another embodiment, at least two of the Fc regions are different. For example, the Fc regions or FcRn binding partners of the proteins of the invention comprise the same number of amino acid residues or they may differ in length by one or more amino acid residues (e.g., by about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet other embodiments, the Fc regions or FcRn binding partners of the protein of the invention may differ in sequence at one or more amino acid positions. For example, at least two of the Fc regions or FcRn binding partners may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).

E) Linkers

The chimeric protein of the present invention further comprises one or more linkers. One type of the linkers is a cleavable linker, which can be cleaved by various proteases when administered to a subject in vivo, e.g., at a site of coagulation. In one embodiment, the cleavable linker allows cleavage of moiety, e.g., a VWF fragment, from the chimeric protein at the site of the coagulation cascade, thus allowing activated FVIII (FVIIIa) to have its FVIIIa activity.

Another type of the linkers is a processable linker, which contains an intracellular cleavage site and thus can be cleaved by an intracellular processing enzyme in a host cell, allowing convenient expression of a polypeptide and formation of a chimeric protein.

One or more linkers can be present between any two proteins in the chimeric protein. In one embodiment, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence by a linker, e.g., a cleavable linker, and the XTEN sequence is further linked to the FVIII protein (i.e., V-L-X-FVIII). In another embodiment, a chimeric protein comprises (i) a VWF fragment, (ii) an

- 1362018203206 08 May 2018 [0242] [0243] [0244]

XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment is linked to the XTEN sequence, and the XTEN sequence is linked to the FVIII protein by a linker, e.g., a cleavable linker (i.e., V-X-L-FVIII).

In certain embodiments, a chimeric protein comprises (i) a VWF fragment, (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof (e.g., a first Fc region), (iv) a FVIII protein, and (v) a second Ig constant region or a portion thereof (e.g., a second Fc region), wherein the VWF fragment is linked to the XTEN sequence by an optional linker, e.g., a cleavable linker. The XTEN sequence can be further linked to the first Ig constant region or a portion thereof by a linker, e.g., a cleavable linker. The FVIII protein (with or without an XTEN sequence) can also be linked to the second Ig constant region or a portion thereof by an optional linker, e.g. a cleavable linker. In certain embodiments, the chimeric protein further comprises one or more linkers, e.g., processable linkers, between the first Ig constant region or a portion thereof (e.g., first Fc region) and the second Ig constant region or a portion thereof (e.g., second Fc region), between the VWF fragment and the second Ig constant region or a portion thereof, or between the FVIII protein and the first Ig constant region or a portion thereof (e.g., first Fc region).

In some embodiments, the present invention includes a chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a first Ig constant region or a portion thereof, and (iv) a second Ig constant region or a portion thereof, wherein the first Ig constant region or a portion thereof and the second Ig constant region or a portion thereof are linked by a processable linker.

The linker useful in the present invention can comprise any organic molecule. In one embodiment, the linker comprises a polymer, e.g., polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker comprises an amino acids sequence. The linker can comprise at least about 10,

20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 ,500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000

- 1372018203206 08 May 2018 [0245] [0246] amino acids. In one embodiment, the linker comprises an XTEN sequence. Additional examples of XTEN can be used according to the present invention and are disclosed in US Patent Publication Nos. 2010/0239554 Al, 2010/0323956 Al, 2011/0046060 Al, 2011/0046061 Al, 2011/0077199 Al, or 2011/0172146 Al, or International Patent Publication Nos. WO 2010091122 Al, WO 2010144502 A2, WO 2010144508 Al, WO 2011028228 Al, WO 2011028229 Al, or WO 2011028344 A2. In another embodiment, the linker is a PAS sequence.

The linker useful in the present invention can comprise any organic molecule. In one embodiment, the linker is a polymer, e.g., polyethylene glycol (PEG) or hydroxyethyl starch (HES). In another embodiment, the linker is an amino acid sequence. The linker can comprise at least about 10, 20, 30, 40, 50,

60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,

1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, or 900-1000 amino acids.

Examples of linkers are well known in the art. In one embodiment, the linker comprises the sequence G_n. The linker can comprise the sequence (GA)_n. The linker can comprise the sequence (GGS)_n. In other embodiments, the linker comprises (GGGS)_n (SEQ ID NO: 57). In still other embodiments, the linker comprises the sequence (GGS)_n(GGGGS)_n (SEQ ID NO: 58). In these instances, n may be an integer from 1-100. In other instances, n may be an integer from 120, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 59), GGSGGSGGSGGSGGG (SEQ ID NO: 60), GGSGGSGGGGSGGGGS (SEQ ID NO: 61), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 62), or GGGGSGGGGSGGGGS (SEQ ID NO: 63). The linker does not eliminate or diminish the VWF fragment activity or the clotting activity of Factor VIII. Optionally, the linker enhances the VWF fragment activity or the clotting activity of Factor VIII protein, e.g., by further diminishing the effects of steric hindrance and making the VWF fragment or Factor VIII portion more accessible to its target binding site.

- 138 2018203206 08 May 2018 [0247] [0248] [0249] [0250] [0251]

In one embodiment, the linker useful for the chimeric protein is 15-25 amino acids long. In another embodiment, the linker useful for the chimeric protein is 15-20 amino acids long. In some embodiments, the linker for the chimeric protein is 10-25 amino acids long. In other embodiments, the linker for the chimeric protein is 15 amino acids long. In still other embodiments, the linker for the chimeric protein is (GGGGS)_n (SEQ ID NO: 64) where G represents glycine, S represents serine and n is an integer from 1-20.

F) Cleavage Sites

The linker may also incorporate a moiety capable of being cleaved either chemically (e.g., hydrolysis of an ester bond), enzymatically (i.e., incorporation of a protease cleavage sequence), or photolytically (e.g., a chromophore such as 3amino-3-(2-nitrophenyl) proprionic acid (ANP)) in order to release one molecule from another.

In one embodiment, the linker is a cleavable linker. The cleavable linkers can comprise one or more cleavage sites at the N-terminus or C-terminus or both. In another embodiment, the cleavable linker consists essentially of or consists of one or more cleavable sites. In other embodiments, the cleavable linker comprises heterologous amino acid linker sequences described herein or polymers and one or more cleavable sites.

In certain embodiments, a cleavable linker comprises one or more cleavage sites that can be cleaved in a host cell (i.e., intracellular processing sites). Non limiting examples of the cleavage site include RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), and RRRRS (SEQ ID NO: 11).

In other embodiments, a cleavable linker comprises one or more cleavage sites that are cleaved by a protease after a chimeric protein comprising the cleavable linker is administered to a subject. In one embodiment, the cleavage site is cleaved by a protease selected from the group consisting of factor XIa, factor Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase2, MMP-12, MMP-13, MMP-17, and MMP-20. In another embodiment, the cleavage site is selected from the group consisting of a FXIa cleavage site (e.g., KLTRjAET (SEQ ID NO: 65)), a FXIa cleavage site (e.g, DFTRjVVG (SEQ ID NO: 66)), a FXIIa cleavage site (e.g., TMTRjIVGG (SEQ ID NO: 67)), a Kallikrein cleavage site (e.g., SPFRjSTGG (SEQ ID NO: 68)), a FVIIa cleavage

- 1392018203206 08 May 2018 [0252] [0253] site (e.g., LQVRjIVGG (SEQ ID NO: 69)), a FIXa cleavage site (e.g., PLGRjIVGG (SEQ ID NO: 70)), a FXa cleavage site (e.g., IEGRjTVGG (SEQ ID NO: 71)), a Flla (thrombin) cleavage site (e.g, LTPRjSLLV (SEQ ID NO: 72)), a Elastase-2 cleavage site (e.g, LGPV jSGVP (SEQ ID NO: 73)), a Granzyme-B cleavage (e.g, VAGDjSLEE (SEQ ID NO: 74)), a MMP-12 cleavage site (e.g., GPAGjLGGA (SEQ ID NO: 75)), a MMP-13 cleavage site (e.g., GPAGjLRGA (SEQ ID NO: 76)), a MMP-17 cleavage site (e.g., APLGjLRLR (SEQ ID NO: 77)), a MMP-20 cleavage site (e.g., PALP jLVAQ (SEQ ID NO: 78)), a TEV cleavage site (e.g., ENLYFQ JG (SEQ ID NO: 79)), a Enterokinase cleavage site (e.g., DDDKjIVGG (SEQ ID NO: 80)), a Protease 3C (PRESCISSION™) cleavage site (e.g., LEVLFQjGP (SEQ ID NO: 81)), and a Sortase A cleavage site (e.g., LPKTjGSES) (SEQ ID NO: 82). In certain embodiments, the FXIa cleavage sites include, but are not limited to, e.g., TQSFNDFTR (SEQ ID NO: 83) and SVSQTSKLTR (SEQ ID NO: 84). Nonlimiting exemplary thrombin cleavage sites include, e.g., DFLAEGGGVR (SEQ ID NO: 85), TTKIKPR (SEQ ID NO: 86), or LVPRG (SEQ ID NO: 87), and a sequence comprising, consisting essentially of, or consisting of ALRPR (SEQ ID NO: 17) (e.g., ALRPRVVGGA (SEQ ID NO: 88)).

In a specific embodiment, the cleavage site is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).

Polynucleotides, Vectors, and Host cells

Also provided in the invention is a polynucleotide encoding (a) a VWF fragment linked to an XTEN sequence and a FVIII protein, (b) a FVIII protein linked to an XTEN sequence and Fc, or (c) a FVIII protein linked to an XTEN sequence and a VWF fragment described herein. When a chimeric protein is a single polypeptide chain (e.g., F2-L2-X-V-L1-FI-FVIII, wherein FVIII comprises a FVIII protein, FI comprises a first Ig constant region or a portion thereof, e.g., a first Fc region, LI comprises a first linker, V comprises a VWF fragment, X comprises an XTEN sequence, L2 comprises a second linker, and F2 comprises a second Ig constant region or a portion thereof, e.g., a second Fc region), the invention is drawn to a single polynucleotide chain encoding the single polypeptide chain. When the chimeric protein comprises a first and a second polypeptide chains (F2-L2-X-V:FVIII-F1), the first polypeptide chain comprising

- 1402018203206 08 May 2018 [0254] a VWF fragment linked to a XTEN sequence, which is further linked to a first Ig constant region or a portion thereof (e.g., a first Fc region) by a cleavable linker (e.g., F2-L2-X-V) and the second polypeptide chain comprising a FVIII protein and a second Ig constant region or a portion thereof (e.g., a second Fc region) (e.g, FVIII-F1), wherein the first polypeptide chain and the second polypeptide chain are associated with each other, a polynucleotide can comprise the first nucleotide sequence and the second nucleotide sequence. In one embodiment, the first polypeptide chain and the second polypeptide chain can be encoded by a single polynucleotide chain. In another embodiment, the first polypeptide chain and the second polypeptide chain are encoded by two different polynucleotides, i.e., a first nucleotide sequence and a second nucleotide sequence. In another embodiment, the first nucleotide sequence and the second nucleotide sequence are on two different polynucleotides (e.g., different vectors). In certain embodiments, the present invention is directed to a set of polynucleotides comprising a first nucleotide chain and a second nucleotide chain, wherein the first nucleotide chain encodes the VWF fragment of the chimeric protein and the second nucleotide chain encodes the FVIII protein. In some embodiments, a chimeric protein comprising two polypeptide chains or three polypeptide chains can be encoded by a single polynucleotide chain, and then processed into two or three (or more) polypeptide chains. In yet other embodiments, a chimeric protein comprising these polypeptide chains can be encoded by two or three polynucleotide chains.

In other embodiments, the set of the polynucleotides further comprises an additional nucleotide chain (e.g., a second nucleotide chain when the chimeric polypeptide is encoded by a single polynucleotide chain or a third nucleotide chain when the chimeric protein is encoded by two polynucleotide chains) which encodes a protein convertase. The protein convertase can be selected from the group consisting of proprotein convertase subtilisin/kexin type 5 (PCSK5 or PC5), proprotein convertase subtilisin/kexin type 7 (PCSK7 or PC5), a yeast Kex 2, proprotein convertase subtilisin/kexin type 3 (PACE or PCSK3), and two or more combinations thereof. In some embodiments, the protein convertase is PACE, PC5, or PC7. In a specific embodiment, the protein convertase is PC5 or PC7.

See International Application no. PCT/US2011/043568.

- 141 2018203206 08 May 2018 [0255] [0256] [0257]

As used herein, an expression vector refers to any nucleic acid construct which contains the necessary elements for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of the invention will include polynucleotides encoding the chimeric protein described herein. In one embodiment, one or more of the coding sequences for the VWF fragment and XTEN, the FVIII protein and XTEN, or both are operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to permit each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5' gene expression sequence results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

A gene expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the coding nucleic acid to which it is operably linked. The gene expression control sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine

- 1422018203206 08 May 2018 [0258] [0259] phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, betaactin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus, and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

In general, the gene expression control sequence shall include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' nontranscribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined coding nucleic acid.

The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.

Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; EpsteinBarr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors well-known in the art. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of

- 143 2018203206 08 May 2018 [0260] [0261] directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, a doublestranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hematopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded within the plasmid. Some

- 1442018203206 08 May 2018 [0262] [0263] [0264] commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using standard molecular biology techniques to remove and/or add specific fragments of DNA.

In one insect expression system that may be used to produce the proteins of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example, the polyhedron gene) of the virus and placed under control of an ACNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (/. e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (see, e.g., Smith et al. (1983) J U/roZ 46:584; U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

Another system which can be used to express the proteins of the invention is the glutamine synthetase gene expression system, also referred to as the GS expression system (Lonza Biologies PLC, Berkshire UK). This expression system is described in detail in U.S. Pat. No. 5,981,216.

In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region

- 145 2018203206 08 May 2018 [0265] [0266] [0267]

El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc Natl

Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may be used.

See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415; Mackett et al.

(1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.

To increase efficiency of production, the polynucleotides can be designed to encode multiple units of the protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with the appropriate enzyme) in order to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, the translation of each polypeptide encoded by the mRNA is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase the yield of polypeptides driven by a single promoter.

Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems, this can include an antibiotic resistance gene such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydro folate reductase (DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. Selectable markers are reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable markers is well within the level of ordinary skill in the art.

Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, U.S. Pat. No. 4,713,339).

- 1462018203206 08 May 2018 [0268] [0269] [0270]

The expression vectors can encode for tags that permit easy purification of the recombinantly produced protein. Examples include, but are not limited to, vector pUR278 (Ruther et al. (1983) EMBO J 2ΆΊ9Ί), in which coding sequences for the protein to be expressed may be ligated into the vector in frame with the lac z coding region so that a tagged fusion protein is produced; pGEX vectors may be used to express proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (thrombin or Factor Xa protease or PRESCISSION PROTEASE™ (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.

The expression vector or vectors are then transfected or co-transfected into a suitable target cell, which will express the polypeptides. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host-expression vector systems may be utilized to express the proteins described herein including both prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g.,

Ti plasmid) containing an appropriate coding sequence; or animal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa, HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell having a definitive nucleus. Eukaryotic cells of animals include cells of vertebrates, e.g., mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells of plants specifically can include,

- 1472018203206 08 May 2018 [0271] [0272] [0273] [0274] [0275] without limitation, yeast cells. A eukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell line. HEK 293 cells are available as CRL-1533 from American Type Culture Collection, Manassas, VA, and as 293-H cells, Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 from Invitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cell is a PER.C6® cell, which is a human cell line derived from retina. PER.C6® cells are available from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from American Type Culture Collection, Manassas, VA. (e.g., CHO-K1; CCL61). In still other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from American Type Culture Collection, Manassas, Va. (e.g., CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell, which is a hybrid cell line of a HEK293 cell and a human B cell line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

In one embodiment, a plasmid including a FVIII(X)-Fc fusion coding sequence, a VWF fragment-L-Fc fusion coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

In another embodiment, a plasmid including a FVIII-Fc fusion coding sequence, a VWF fragment-XTEN-L-Fc fusion coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

In other embodiments, a plasmid including a FVIII(X)-Fc fusion coding sequence, a Fc coding sequence, or both and a selectable marker, e.g., zeocin resistance, are transfected into HEK 293 cells, for production of a chimeric protein.

In some embodiments, a first plasmid including a FVIII(X)-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a

- 148 2018203206 08 May 2018 [0276] [0277] [0278] second plasmid including an Fc coding sequence or a VWF fragment-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

In still other embodiments, a first plasmid including a FVIII-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a VWF fragment-XTEN-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

In yet other embodiments, a first plasmid including a FVIII(X)-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a VWF fragment-XTEN-L-Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

In certain embodiments, a first plasmid, including a chimeric protein encoding FVIII (with or without XTEN)-F1-L1-V-XTEN-L2-F2 coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a protein convertase coding sequence and a second selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of the chimeric protein. The promoters for the FVIII(X)-Fc coding sequence and the VWF-XTEN-Fc coding sequence can be different or they can be the same.

- 1492018203206 08 May 2018 [0279] [0280] [0281]

In still other embodiments, transfected cells are stably transfected. These cells can be selected and maintained as a stable cell line, using conventional techniques known to those of skill in the art.

Host cells containing DNA constructs of the protein are grown in an appropriate growth medium. As used herein, the term appropriate growth medium means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally, the media can contain one or more selection factors. Optionally the media can contain bovine calf serum or fetal calf serum (FCS). In one embodiment, the media contains substantially no IgG. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM/F12). In one embodiment, the medium is CD293 (Invitrogen, Carlsbad, CA.). In another embodiment, the medium is CD 17 (Invitrogen, Carlsbad, CA.). Selection of a medium appropriate for the particular cell line used is within the level of those ordinary skilled in the art.

In order to co-express the two polypeptide chains of the chimeric protein, the host cells are cultured under conditions that allow expression of both chains. As used herein, culturing refers to maintaining living cells in vitro for at least a definite time. Maintaining can, but need not include, an increase in population of living cells. For example, cells maintained in culture can be static in population, but still viable and capable of producing a desired product, e.g., a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include appropriate selection of culture media, media supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing can include the use of any of various types of scale-up systems including shaker flasks, roller bottles, hollow fiber bioreactors, stirred-tank bioreactors, airlift bioreactors, Wave bioreactors, and others.

- 1502018203206 08 May 2018 [0282] [0283] [0284] [0285] [0286] [0287]

The cell culture conditions are also selected to allow association of the VWF fragment with the FVIII protein. Conditions that allow expression of the VWF fragment and/or the FVIII protein may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 media (Invitrogen, Carlsbad, CA) or OptiCHO media (Invitrogen, Carlsbad, CA) supplemented with 4 mM glutamine.

In one aspect, the present invention is directed to a method of expressing, making, or producing the chimeric protein of the invention comprising a) transfecting a host cell comprising a polynucleotide encoding the chimeric protein and b) culturing the host cell in a culture medium under a condition suitable for expressing the chimeric protein, wherein the chimeric protein is expressed.

In further embodiments, the protein product containing the VWF fragment linked to an XTEN sequence or the FVIII protein linked to an XTEN sequence is secreted into the media. Media is separated from the cells, concentrated, filtered, and then passed over two or three affinity columns, e.g., a protein A column and one or two anion exchange columns.

In certain aspects, the present invention relates to the chimeric protein produced by the methods described herein.

In vitro production allows scale-up to give large amounts of the desired altered polypeptides of the invention. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography (HIC, chromatography over DEAE-cellulose or affinity chromatography.

Pharmaceutical Composition

Compositions containing the chimeric protein of the present invention may contain a suitable pharmaceutically acceptable carrier. For example, they may contain excipients and/or auxiliaries that facilitate processing of the active compounds into preparations designed for delivery to the site of action.

- 151 2018203206 08 May 2018 [0288] [0289] [0290] [0291]

The pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous, subcutaneous, or intramuscular) by bolus injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., pyrogen free water.

Suitable formulations for parenteral administration also include aqueous solutions of the active compounds in water-soluble form, for example, watersoluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and dextran. Optionally, the suspension may also contain stabilizers. Liposomes also can be used to encapsulate the molecules of the invention for delivery into cells or interstitial spaces. Exemplary pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like. In some embodiments, the composition comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. In other embodiments, the compositions comprise pharmaceutically acceptable substances such as wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the active ingredients.

Compositions of the invention may be in a variety of forms, including, for example, liquid (e.g., injectable and infusible solutions), dispersions, suspensions, semi-solid and solid dosage forms. The preferred form depends on the mode of administration and therapeutic application.

The composition can be formulated as a solution, micro emulsion, dispersion, liposome, or other ordered structure suitable to high drug

- 1522018203206 08 May 2018 [0292] [0293] [0294] concentration. Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterilefiltered solution. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

The active ingredient can be formulated with a controlled-release formulation or device. Examples of such formulations and devices include implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations and devices are known in the art. See e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Injectable depot formulations can be made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the polymer employed, the rate of drug release can be controlled. Other exemplary biodegradable polymers are polyorthoesters and polyanhydrides. Depot injectable formulations also can be prepared by entrapping the drug in liposomes or microemulsions.

Supplementary active compounds can be incorporated into the compositions. In one embodiment, the chimeric protein of the invention is formulated with another clotting factor, or a variant, fragment, analogue, or derivative thereof For example, the clotting factor includes, but is not limited to,

- 153 2018203206 08 May 2018 [0295] [0296] [0297] [0298] factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor

XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue factor (rsTF) or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-aminocaproic acid, tranexamic acid.

Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa. 1980).

In addition to the active compound, the liquid dosage form may contain inert ingredients such as water, ethyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan.

Non-limiting examples of suitable pharmaceutical carriers are also described in Remington's Pharmaceutical Sciences by E. W. Martin. Some examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can include suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents.

- 1542018203206 08 May 2018 [0299] [0300] [0301] [0302]

Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

For buccal administration, the composition may take the form of tablets or lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or nebulizer, with optionally a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In one embodiment, a pharmaceutical composition comprises a chimeric protein, the polynucleotide encoding the chimeric protein, the vector comprising the polynucleotide, or the host cell comprising the vector, and a pharmaceutically acceptable carrier. The FVIII protein in a chimeric protein has extended half-life compared to wild type FVIII protein or the corresponding FVIII protein without the VWF fragment. In one embodiment, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII. In another embodiment, the half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at

- 155 2018203206 08 May 2018 [0303] [0304] [0305] least about 60 hours, at least about 72 hours, at least about 84 hours, at least about hours, or at least about 108 hours.

In some embodiments, the composition is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration. The parenteral administration can be intravenous or subcutaneous administration.

In other embodiments, the composition is used to treat a bleeding disease or condition in a subject in need thereof. The bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof. In still other embodiments, the subject is scheduled to undergo a surgery. In yet other embodiments, the treatment is prophylactic or ondemand.

Gene Therapy

A chimeric protein thereof of the invention can be produced in vivo in a mammal, e.g., a human patient, using a gene therapy approach to treatment of a bleeding disease or disorder selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath would be therapeutically beneficial. In one embodiment, the bleeding disease or disorder is hemophilia. In another embodiment, the bleeding disease or disorder is hemophilia A. This involves administration of a suitable chimeric protein-encoding nucleic acid operably linked to suitable expression control sequences. In certain embodiment, these sequences are incorporated into a viral vector. Suitable viral vectors for such gene

- 1562018203206 08 May 2018 [0306] [0307] [0308] [0309] therapy include adenoviral vectors, lentiviral vectors, baculoviral vectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viral vectors, herpes simplex viral vectors, and adeno associated virus (AAV) vectors. The viral vector can be a replication-defective viral vector. In other embodiments, an adenoviral vector has a deletion in its El gene or E3 gene. When an adenoviral vector is used, the mammal may not be exposed to a nucleic acid encoding a selectable marker gene. In other embodiments, the sequences are incorporated into a non-viral vector known to those skilled in the art.

Methods of Using Chimeric Protein

The present invention is directed to a method of using a chimeric protein described herein to prevent or inhibit endogenous VWF binding to a FVIII protein. The present invention is also directed to a method of using a chimeric protein having a FVIII protein linked to XTEN and an Ig constant region or a portion thereof.

One aspect of the present invention is directed to preventing or inhibiting FVIII interaction with endogenous VWF by blocking or shielding the VWF binding site on the FVIII from endogenous VWF and at the same time extending half-life of the FVIII protein using an XTEN sequence in combination with an Ig constant region or a portion thereof, which can also be a half-life extender. In one embodiment, the invention is directed to a method of constructing a FVIII protein having half-life longer than wild-type FVIII. In one embodiment, an XTEN sequence inhibits or prevents interaction of a FVIII protein in a chimeric protein with endogenous VWF. In another embodiment, an Ig constant region or a portion thereof inhibits or prevents interaction of the FVIII protein with endogenous VWF. The chimeric protein useful in the method includes any one or more chimeric protein described herein.

Another aspect of the invention includes a method of administering to a subject in need thereof a chimeric protein comprising a FVIII protein having halflife longer than wild-type FVIII, wherein the method comprises administering the chimeric protein described herein to the subject.

In one embodiment, the invention is directed to a method of using an XTEN sequence and an Ig constant region or a portion thereof to extend a half-life of a FVIII protein and a VWF fragment to prevent or inhibit endogenous VWF

- 1572018203206 08 May 2018 [0310] [0311] [0312] interaction with a FVIII protein. A FVIII protein linked to an XTEN sequence (e.g., FVIII(X)) and then bound to or associated with a VWF fragment is shielded or protected from the clearance pathway of VWF and thus has reduced clearance compared to the FVIII protein not bound to the VWF fragment. The shielded FVIII protein thus has maximum extension of a half-life compared to a FVIII protein not bound to or associated with the XTEN sequence and the VWF fragment. In certain embodiments, the FVIII protein associated with or protected by a VWF fragment and linked to an XTEN sequence is not cleared by a VWF clearance receptor. In other embodiments, the FVIII protein associated with or protected by a VWF fragment and linked to an XTEN sequence is cleared from the system slower than the FVIII protein that is not associated with or protected by the VWF fragment and linked to the XTEN sequence.

In one aspect, the chimeric protein comprising the FVIII protein linked to an XTEN sequence or the FVIII protein bound to or associated with a VWF fragment linked to XTEN has reduced clearance from circulation as the VWF fragment does not contain a VWF clearance receptor binding site. The VWF fragment prevents or inhibits clearance of FVIII bound to or associated with the VWF fragment from the system through the VWF clearance pathway. The VWF fragments useful for the present invention can also provide at least one or more VWF-like FVIII protection properties that are provided by endogenous VWF. In certain embodiments, the VWF fragment or the XTEN sequence can also mask one or more FVIII clearance receptor binding site, thereby preventing clearance of FVIII by its own clearance pathway.

In some embodiments, the prevention or inhibition of a FVIII protein binding to endogenous VWF by the VWF fragment or the XTEN sequence can be in vitro or in vivo.

Also provided is a method of increasing the half-life of a FVIII protein comprising administering the chimeric protein described herein to a subject in need thereof. The half-life of non-activated FVIII bound to or associated with full-length VWF is about 12 to 14 hours in plasma. In VWD type 3, wherein there is almost no VWF in circulation, the half-life of FVIII is only about six hours, leading to symptoms of mild to moderate hemophilia A in such patients due to decreased concentrations of FVIII. The half-life of the FVIII protein linked to or

- 158 2018203206 08 May 2018 [0313] [0314] associated with the VWF fragment or the XTEN sequence of the present invention can increase at least about 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.6 times, 2.7. times, 2.8 times,

2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, or 4.0 times higher than the half-life of the nonactivated FVIII bound to or associated with full-length VWF.

In one embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or linked to an Ig constant region or a portion thereof in the chimeric protein comprising an XTEN sequence increases at least about 2 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 7 times, 8 times, 9 times, or 10 times higher than the half-life of the non-activated FVIII bound to or associated with full-length VWF. In another embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or an Ig constant region or a portion thereof in the chimeric protein comprising an XTEN sequence increases about 2 to about 5 times, about 3 to about 10 times, about 5 to about 15 times, about 10 to about 20 times, about 15 to about 25 times, about 20 to about 30 times, about 25 to about 35 times, about 30 to about 40 times, about 35 to about 45 times higher than the half-life of the nonactivated FVIII bound to or associated with full-length VWF or wild type FVIII.

In a specific embodiment, the half-life of the FVIII protein linked to or associated with the VWF fragment or linked to an Ig constant region in the chimeric protein comprising an XTEN sequence increases at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 times higher than the half-life of the wild type FVIII in a FVIII and VWF double knockout mouse.

In some embodiments, the half-life of the chimeric protein comprising the VWF fragment fused to a first Ig constant region or a portion thereof, e.g., a first Fc region and an XTEN sequence, and a FVIII protein linked to an XTEN sequence and a second Ig constant region or a portion thereof, e.g., a second Fc region, is longer than the half-life of a FVIII associated with endogenous VWF.

In other embodiments, the half-life of the chimeric protein is at least about 1.5 times, 2 times, 2.5 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.5 times, or 5.0 times the half-life of wild type FVIII or a FVIII protein associated with endogenous VWF.

- 1592018203206 08 May 2018 [0315]

In some embodiments, as a result of the invention the half-life of the FVIII protein is extended compared to a FVIII protein without the VWF fragment or wild-type FVIII. The half-life of the chimeric protein of the invention is at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than the half-life of a FVIII protein without the VWF fragment or wild-type FVIII. In one embodiment, the half-life of FVIII is about 1.5-fold to about 20-fold, about 1.5 fold to about 15 fold, or about 1.5 fold to about 10 fold longer than the half-life of wild-type FVIII. In another embodiment, the half-life of the FVIII is extended about 2-fold to about 10-fold, about 2-fold to about 9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold, about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2fold to about 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold, about 2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about 2.5-fold to about 5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3-fold, about 3-fold to about 10fold, about 3-fold to about 9-fold, about 3-fold to about 8-fold, about 3-fold to about 7-fold, about 3-fold to about 6-fold, about 3-fold to about 5-fold, about 3fold to about 4-fold, about 4-fold to about 6 fold, about 5-fold to about 7-fold, or about 6-fold to about 8 fold as compared to wild-type FVIII or a FVIII protein without the VWF fragment. In other embodiments, the half-life of the chimeric protein of the invention is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours. In still other embodiments, the half-life of the chimeric protein of the invention is about 15 hours to about two weeks, about 16 hours to about one week, about 17 hours to about one week, about 18 hours to about one week, about 19 hours to

- 1602018203206 08 May 2018 [0316] [0317] [0318] about one week, about 20 hours to about one week, about 21 hours to about one week, about 22 hours to about one week, about 23 hours to about one week, about 24 hours to about one week, about 36 hours to about one week, about 48 hours to about one week, about 60 hours to about one week, about 24 hours to about six days, about 24 hours to about five days, about 24 hours to about four days, about 24 hours to about three days, or about 24 hours to about two days.

In some embodiments, the average half-life of the chimeric protein of the invention per subject is about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours, about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours (4 days), about 108 hours, about 120 hours (5 days), about six days, about seven days (one week), about eight days, about nine days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

In addition, the invention provides a method of treating or preventing a bleeding disease or disorder comprising administering an effective amount of a chimeric protein. In one embodiment, the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intraabdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath. In a specific embodiment, the bleeding disease or disorder is hemophilia A.

The chimeric protein comprising an XTEN sequence and an Ig constant region or a portion thereof in combination with a VWF fragment described herein, that prevents or inhibits interaction of the FVIII protein with endogenous VWF prepared by the invention, has many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general

- 161 2018203206 08 May 2018 [0319] [0320] [0321] [0322] hemostatic agent. In one embodiment, the invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of the chimeric protein.

The FVIII protein portion in the chimeric protein treats or prevents a hemostatic disorder by serving as a cofactor to Factor IX on a negatively charged phospholipid surface, thereby forming a Xase complex. The binding of activated coagulation factors to a phospholipid surface localizes this process to sites of vascular damage. On a phospholipid surface, Factor Villa increases the maximum velocity of Factor X activation by Factor IXa, by approximately 200,000-fold, leading to the large second burst of thrombin generation.

The chimeric protein of the invention can be used to treat any hemostatic disorder. The hemostatic disorders that may be treated by administration of the chimeric protein of the invention include, but are not limited to, hemophilia A, as well as deficiencies or structural abnormalities relating to Factor VIII. In one embodiment, the hemostatic disorder is hemophilia A.

The chimeric protein of the invention can be used prophylacticaliy to treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand's factor. In one embodiment, the hemostatic disorder is an inherited disorder. In another embodiment, the hemostatic disorder is an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an auto-immune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).

The invention also relates to methods of treating a subject that does not have a congenital hemostatic disorder, but has a secondary disease or condition resulting in acquisition of a hemostatic disorder, e.g., due to development of an anti-FVIII antibody or a surgery. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of the chimeric protein prepared by the present methods.

- 1622018203206 08 May 2018 [0323] [0324] [0325] [0326] [0327]

The present invention is also related to methods of reducing immunogenicity of FVIII or inducing less immunogenicity against FVIII comprising administering an effective amount of the chimeric proteins described herein, or the polynucleotides encoding the same.

In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The chimeric protein of the invention can be administered prior to, during, or after surgery as a prophylactic regimen. The chimeric protein of the invention can be administered prior to, during, or after surgery to control an acute bleeding episode..

The chimeric protein of the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding. Non limiting examples of bleeding episodes include a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath, and any combinations thereof.

In prophylactic applications, one or more compositions containing the chimeric protein of the invention or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance or reduce symptoms associated with a disease or disorder. Such an amount is defined to be a prophylactic effective dose. In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of polypeptide per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmuno conjugates and higher doses for cytotoxin-drug modified polypeptides) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

In some embodiments, a chimeric protein or a composition of the invention is used for on-demand treatment, which includes treatment for a bleeding episode,

- 163 2018203206 08 May 2018 [0328] [0329] hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis (head trauma), gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, or bleeding in the illiopsoas sheath. The subject may be in need of surgical prophylaxis, peri-operative management, or treatment for surgery. Such surgeries include, e.g., minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intra-abdominal surgery, intrathoracic surgery, or joint replacement surgery.

In one embodiment, the chimeric protein of the present invention is administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route. The chimeric protein comprising a VWF fragment and a FVIII protein of the present invention can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the site of bleeding or implanted into bandage/dressing. The dose of the chimeric protein will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 pg/kg body weight. In one embodiment, the dosing range is 0.1-1,000 pg/kg. In another embodiment, the dosing range is 0.1-500 pg/kg. The protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e.g., STA-CLOT VIIa-rTF clotting assay or ROTEM clotting assay. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. All patents, publications, and articles referred to herein are expressly and specifically incorporated herein by reference.

- 1642018203206 08 May 2018 [0330] [0331] [0332] [0333]

Examples

Throughout the examples, the following materials and methods were used unless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, biophysics, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in electrophoresis. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., CS.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992). Example 1: Cloning different VWF domains (Figure 1) (a) Cloning of pSYN-VWF-002 pSYN-VWF-002 contains nucleotide sequences encoding a VWF fragment, which are amino acids 1-477 of SEQ ID NO: 100. [VWF-D'D3 protein sequence] Amino acid numbering represents the mature VWF sequence without propeptide and corresponds to amino acids 764-1240 of SEQ ID NO: 2. pSYNVWF-002 construct has the FVIII signal peptide at N-terminus, which allows proper secretion of the synthesized protein and followed by a 6xHis tag at Cterminus, which is used for protein purification. It was synthesized by using following primer combinations:

ESC48- Fwd - VWF-DO3 with VIII signal and BsiWl site

TCGCGACGTACGGCCGCCACCATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGT GCCTTTTGCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATG (SEQ ID NO: 90)

ESC51- Rev- VWF D’D3 (1-477 amino acid) with 6His and Not 1 site

TGACCTCGAGCGGCCGCTCAGTGGTGATGGTGATGATGCGGCTCCTGGCAGGCTT CACAGGTGAGGTTGACAAC (SEQ ID NO: 91)

A 50 μΐ PCR reaction was carried out with ESC 48/ESC 51 primer combinations and full length VWF plasmid as the template, using the 2 step PCR

- 165 2018203206 08 May 2018 [0334] [0335] [0336] amplification cycle: 94 °C 2minutes; 21 cycles of (96 °C 30 seconds, 68 °C 2 minute). The 1460bp band was gel purified with a Gel Extraction kit (Qiagen,

Valencia, Calif.) and cloned into the BsiWI and Notl restriction sites of pcDNA 4 to generate pSYN-VWF 002.

(b) Cloning of pSYN-VWF- 010 and 013 pSYN-VWF-010 was constructed using pSYN-VWF-008 and pSYNVWF-002. pSYN-VWF-008 contains the full-length VWF sequence in pcDNA 3.1 (amino acids 1-2813 of SEQ ID NO: 2), it includes 763 amino acid propeptide (i.e., D1D2 domains) followed by remaining 2050 amino acids sequence of mature VWF. The FVIII signal peptide in pSYN-VWF-002 was replaced with D1D2 domains from pSYN-VWF-008, the resulting construct is pSYN-VWF-010. pSYN-VWF- 008 has a BamHl site at Arg907 and Notl site at the end of coding region (after stop codon). pSYN- VWF- 008 and 002 were digested with BamHl and Notl restriction enzymes. Inserts from pSYN- VWF-002 (1026 bp) were ligated into bamHl/Notl digested pSYN-VWF- 008 (8242bp) to obtain pSYNVWF -010 (D1D2DO3: amino acid 1-1240 of SEQ ID NO: 2), a 6xHis tag was also added at the C-terminus. In transformed cells pSYN-VWF-010 is synthesized with propeptide but due to intracellular processing the secreted products do not contain any propeptide (D1D2). Protein from VWF-010 exists as dimer.

pSYN-VWF-010 was used to generate pSYN-VWF-013 which has two point mutations at C336A and C379A corresponding to SEQ ID NO: 100 (amino acid numbering represents mature VWF sequence without D1D2 domain-VWF sequence 2). These mutations are predicted to prevent dimerization of VWF D'D3 domain.

(c) Cloning of pSYN-VWF-025 and pSYN-VWF-029 pSYN-VWF-025 contains wild type D1D2D'D3 sequences of full-length

VWF in pLIVE vector, and pSYN-VWF-029 contains D1D2DO3 sequence with C336A and C379A mutation. For cloning pSYN-VWF-025, the following primer combination was used:

ESC 89-fwd withNhelsite= CTCACTATAGGGAGACCCAAGCTGGCTAGCCG (SEQ ID NO: 92)

- 1662018203206 08 May 2018 [0337] [0338] [0339] [0340]

ESC 91-rev with Sail=

CTGGATCCCGGGAGTCGACTCGTCAGTGGTGATGGTGATGATG (SEQ ID NO:

93)

A 50 μΐ PCR reaction was carried out with ESC 89/ESC91 primer combinations and either pSYN-VWF 010 ( for pSYN-VWF-025) or pSYN-VWF 013 ( for pSYN-VWF-029) plasmid as the template using the 3 step PCR amplification cycle: 94 °C 2minutes; 21 cycles of (96 °C -30 seconds, 55 °C-30 second, 68 °C-4 minutes). The expected sized band (~3800bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, Calif.) and cloned into the Nhe land Sail restriction sites of pLIVE-Mirus vector (Invitrogen, Carlsbad, Calif.) to generate pSYN-VWF 025 and 029.

(d) Cloning pSYN-VWF-031 pSYN-VWF-031 is a D1D2D'D3(C336A/C379A) -Fc construct which has a 48 amino acid long thrombin cleavable linker (8x GGGGS (SEQ ID NO 94) + thrombin site) in between the VWF D1D2DD3(C336A/C379A) and the Fc sequences. To make this construct, VWF-Fc region was amplified from construct pSYN-FVIII-064 (refer FVIII-VWF construct below). pSYN-FVIII-VWF was digested with Xbal and Nhel. Resulting insert region of 4165bp, containing the VWF fragment and Fc region was used as a template for amplifying the VWF and Fc region by primer combinations LW 22/LW23.

LW 22-FWD-VWF-DD3 with FVIII signal sequence and BsiWl site

GCGCCGGCCGTACGATGCAAATAGAGCTCTCCACCTGCTTCTTTCTGTGCCTTTT GCGATTCTGCTTTAGCCTATCCTGTCGGCCCCCCATG (SEQ ID NO: 95)

LW 23-Rev- Fc with stop codon andNotl site

TCATCAATGTATCTTATCATGTCTGAATTCGCGGCCGCTCATTTACC (SEQ ID NO:96)

The PCR product obtained from LW22/LW23 amplification (~2300bp) was cloned in BsiWl/Notl digested pSYN-VWF-002 to obtain pSYN-VWF-014 intermediate. pSYN-VWF-014 contains FVIII signal peptide-DD3-20 amino acid thrombin cleavable linker followed by the Fc region.

To generate the D1D2D’D3-Fc construct, the D1D2DO3 region was amplified from pSYN-VWF-013 using primer combination LW24/LW27 by standard PCR method.

LW24- Fwd- VWF D1D2DO3 cloning oligo with BsiWl site

- 1672018203206 08 May 2018

GCGCCGGCCGTACGATGATTCCTGCCAGATTTGCCGGGGTG (SEQ ID NO:97)

LW27-Rev-VWF D'D3 oligo with EcoRV

CCACCGCCAGATATCGGCTCCTGGCAGGCTTCACAGGTGAG (SEQ ID NO:98) [0341] The PCR product obtained from LW22/LW23 amplification (~3750bp) was cloned in BsiWl/EcoRV digested pSYN-VWF-014 to obtain pSYN-VWF015 intermediate. The linker length between the VWF fragment and Fc region was changed to obtain pSYN-VWF-031.

VWF-D1D2DO3 protein sequence 1 (SEQ ID NO: 99)

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

- 168

2018203206 08 May 2018 [0342]

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE

KVCGLCGNFD

L001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD

SSPATCHNNI

LG51 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCACF

L101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY ECEWRYNSCA

L151 PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE

L.2 01 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP* VWF-D'D3 protein sequence 2 (SEQ ID NO: 100)

SLSCRPPMVK LVCPADNLRA EGLECTKTCQ NYDLECMSMG CVSGCLCPPG

MVRHENRCVA LERCPCFHQG KEYAPGETVK IGCNTCVCRD RKWNCTDHVC

101 DATCSTIGMA HYLTFDGLKY LFPGECQYVL VQDYCGSNPG TFRILVGNKG

151 CSHPSVKCKK RVTILVEGGE IELFDGEVNV KRPMKDETHF EVVESGRYII

201 LLLGKALSVV WDRHLSISVV LKQTYQEKVC GLCGNFDGIQ NNDLTSSNLQ

251 VEEDPVDFGN SWKVSSQCAD TRKVPLDSSP ATCHNNIMKQ TMVDSSCRIL

301 TSDVFQDCNK LVDPEPYLDV CIYDTCSCES IGDCACFCDT IAAYAHVCAQ

351 HGKVVTWRTA TLCPQSCEER NLRENGYECE WRYNSCAPAC QVTCQHPEPL

401 ACPVQCVEGC HAHCPPGKIL DELLQTCVDP EDCPVCEVAG RRFASGKKVT

451 LNPSDPEHCQ ICHCDVVNLT CEACQEP

Example 2: Effects of D'D3 and XTEN fusion on FVIII half-life extension

To evaluate D'D3 FVIII half-life extension potential on rFVIII-XTEN fusion protein, a VWF D'D3 dimer was introduced into FVIII-VWF DKO mice by hydrodynamic injection of its corresponding DNA construct VWF-025 (Example 1). After D'D3 has reached the steady state expression (day5 post injection), a single dose of rFVIII-XTEN was administered by IV injection at 200 IU/kg dose. Blood samples were collected up to 120hrs post rFVIII-XTEN dosing. Plasma FVIII activity was analyzed by a FVIII chromogenic assay. The D'D3 expression level was measured by VWF ELISA, and rFVIIIFc PK profile was analyzed using WinNonlin program.

The study results were shown in Figure 2, and the PK parameter of rFVIIIXTEN with/without D'D3 in circulation was listed in Table 16. The D'D3 dimer further extended rFIII-XTEN ti/2 from 3.4hr to 17.8hr, a 5 fold increase. In [0343]

- 169addition to half-life, 5 fold of increase on MRT, 3.6 fold increases on AUC, 3.8 fold decreases on clearance were also observed.

[0344] We have observed a synergistic effect of D'D3 fragment and XTEN technology, a serial of FVIII/VWF/ΧΤΕΝ constructs will be evaluated for their

FVIII half-life extension potential in Hemophilic animals.

TABLE 16: rFVIII-XTEN PK parameter with/without D'D3 in blood

2018203206 08 May 2018 circulation

Treatment	5min Recovery (%)	^1/2 (hr)	MRT (hr)	Cl (mL/hr/kg)	Vss (mL/kg)	AUC D (hr*kg*mIU /mL/mIU)
rFVIIIXTE N VWF-025	80	17.8	19.3	3.5	67.4	0.29
rFVIIIXTE N	74	3.4	3.8	13.1	63.68	0.08
Improveme nt fold	1.1	5.2	5.1	3.8	0.9	3.6

Protein purification of FVIII-XTEN [0345] An AE288 XTEN was inserted at the C-terminus of BDD-FVIII for this study. To purify this protein, a tangential flow filtration (TFF) step was used first to buffer exchange the conditioned media. Products in the filtrate were then captured using a strong anion exchange chromatography, and then further purified using affinity chromatography. Purity of the molecule was acceptable by HPLCSEC and was further confirmed by western blotting. The specific activity of the molecule was comparable to B-domain deleted FVIII, as measured by aPTT assay and ELISA.

FVIII chromogenic assay [0346] The FVIII activity was measured using the COATEST SP FVIII kit from

DiaPharma (lot# N089019) and all incubations were performed on a 37°C plate heater with shaking.

[0347] The range of rFVIII standard was from 100 mlU/mL to 0.78 mlU/mL. A pooled normal human plasma assay control and plasma samples (diluted with IX Coatest buffer) were added into Immulon 2HB 96-well plates in duplicate (25 pL/well). Freshly prepared IXa/FX/Phospholipid mix (50 pL), 25 pL of 25mM CaC12, and 50 pL of FXa substrate were added sequentially into each well with 5

- 1702018203206 08 May 2018 [0348] [0349] [0350] minutes incubation between each addition. After incubating with the substrate, 25 pL of 20% Acetic Acid was added to terminate the color reaction, and the absorbance of OD405 was measured with a SpectraMAX plus (Molecular

Devices) instrument. Data were analyzed with SoftMax Pro software (version

5.2). The Lowest Level of Quantification (LLOQ) is 7.8 mlU/mL.

VWF ELISA:

Goat anti-human VWF antibody (Affinity purified, affinity biological, GAVWF-AP) was used as the capture antibody at 0.5ug/well and VWF-EIA-D (Affinity Biologicals, VWF-EIA-D, 1:100 dilution) was used as the detecting antibody for the VWF ELISA. ELISA assay was performed following the standard ELISA procedure, TMB was used as the HRP substrate, PBST/1.5% BSA/0.5M NaCl buffer was used as blocking and binding buffer. The assay standard range is lOOng to 0.78ng, and assay's lowest limit of quantification (LLOQ) is 7.8ng/mL.

Example 3: Plasmid construction of XTEN containing FVIII/VWF constructs (a) Cloning of pSYN-FVIII-161 (Figure 3)

The FVIII-161 plasmid comprises a single chain Fc (scFc) scaffold with enzyme cleavage sites which are processed during synthesis in a cell. The construct has a FVIII binding domain of full-length VWF ( DD3).

Plasmid (pSYN-FVIII-161) was designed for the expression FVIII-Fc and VWF-Fc heterodimer, where the D’D3 domains to bind FVIII and prevents FVIII interaction with phospholipids and activated protein C. Protein from pSYN-FVIII161 is expressed in the cell as a single polypeptide where the C-terminus of the FVIII-Fc subunit is linked to the N-terminus of the VWF D’D3-Fc subunit by a 6x (GGGGS) polypeptide linker (SEQ ID NO: 64). In addition, RRRRS (SEQ ID NO: 11) and RKRRKR (SEQ ID NO: 10) sequences were inserted at the 5' and 3' end of the polypeptide linker, respectively, for intracellular cleavage by proprotein convertases following the last Arg at each sequence. Hence, the cells can express a double chain FVIII-Fc/D'D3-Fc heterodimer where the FVIII-Fc chain has a RRRRS sequence (SEQ ID NO: 11) at the C-terminus, but the remainder of the linker sequence has been removed. An AE288 XTEN fragment immediately followed by IS{5X(GGGGS)}LVPRGSGG (SEQ ID NO: 122) polypeptide (contains thrombin cleavage site) is introduced in between the VWF domains and

- 171 2018203206 08 May 2018 [0351] the Fc region to facilitate release of the VWF fragment from FVIII once the FVIII

VWF hetero-dimeric protein is activated by thrombin allowing interaction of

FVIII with other clotting factors.

pSYN-FVIII-161 (SEQ ID NO: 101).protein sequence (FVIII sequence amino acid position 1-1457; underlined region represents Fc region; curvy underline represents cleavable linker in between first Fc and VWF fragment; double underlined region represents VWF fragment; bold region represents cleavable linker in between VWF fragment and Fc.

1 MQIELSTCFF GELPVDARFP	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQSDL
51 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
101 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
151 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
201 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
251 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
301 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
351 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
401 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
451 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
501 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
551 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
601 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
651 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
701 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
751 SKNNAIEPRS DTISVEMKKE	FSQNPPVLKR	HQREITRTTL	QSDQEEIDYD
601 DFDIYDEDEN HVLRNRAQSG	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
851 SVPQFKKVVF EVEDNIMVTF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
901 RNQASRPYSF FWKVQHHMAP	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY

- 172

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951 TKDEFDCKAW AHGRQVTVQE	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
1001 FALFFTIFDE	TKSWYFTENM	ERNCRAPCNI	QMEDPTFKEN
YRFHAINGYI 1051 MDTLPGLVMA	QDQRIRWYLL	SMGSNENIHS	IHFSGHVFTV
RKKEEYKMAL 1101 YNLYPGVFET	VEMLPSKAGI	WRVECLIGEH	LHAGMSTLFL
VYSNKCQTPL 1151 GMASGHIRDF	QITASGQYGQ	WAPKLARLHY	SGSINAWSTK
EPFSWIKVDL 1201 LAPMIIHGIK	TQGARQKFSS	LYISQFIIMY	SLDGKKWQTY
RGNSTGTLMV 1251 FFGNVDSSGI	KHNIFNPPII	ARYIRLHPTH	YSIRSTLRME
LMGCDLNSCS 1301 MPLGMESKAI	SDAQITASSY	FTNMFATWSP	SKARLHLQGR
SNAWRPQVNN 1351 PKEWLQVDFQ	KTMKVTGVTT	QGVKSLLTSM	YVKEFLISSS
QDGHQWTLFF 14 01 QNGKVKVFQG	NQDSFTPVVN	SLDPPLLTRY	LRIHPQSWVH
QIALRMEVLG 1451 CEAQDLYDKT	HTCPPCPAPE	LLGGPSVFLF	PPKPKDTLMI
SRTPEVTCVV 1501 VDVSHEDPEV	KFNWYVDGVE	VHNAKTKPRE	EQYNSTYRVV
SVLTVLHQDW 1551 LNGKEYKCKV	SNKALPAPIE	KTISKAKGQP	REPQVYTLPP
SRDELTKNQV 1601 SLTCLVKGFY	PSDIAVEWES	NGQPENNYKT	TPPVLDSDGS
FFLYSKLTVD 1651 KSRWQQGNVF	SCSVMHEALH	NHYTQKSLSL	SPGKRRRRSG
GGGSGGGGSG 17 01 GGGSGGGGSG	GGGSGGGGSR	KRRKRSLSCR	PPMVKLVCPA
DNLRAEGLEC 1751 TKTCONYDLE	CMSMGCVSGC	LCPPGMVRHE	NRCVALERCP
CFHQGKEYAP 1801 GETVKIGCNT	CVCRDRKWNC	TDHVCDATCS	TIGMAHYLTF
DGLKYLFPGE 1851 CQYVLVQDYC	GSNPGTFRIL	VGNKGCSHPS	VKCKKRVTIL
VEGGEIELFD 1901 GEVNVKRPMK	DETHFEVVES	GRYIILLLGK	ALSVVWDRHL
SISVVLKQTY 1951 OEKVCGLCGN	FDGIONNDLT	SSNLOVEEDP	VDFGNSWKVS
SOCADTRKVP 2001 LDSSPATCHN	NIMKQTMVDS	SCRILTSDVF	QDCNKLVDPE
PYLDVCIYDT 2051 CSCESIGDCA	AFCDTIAAYA	HVCAQHGKVV	TWRTATLCPQ
SCEERNLREN 2101 GYEAEWRYNS	CAPACOVTCO	HPEPLACPVO	CVEGCHAHCP
PGKILDELLO 2151 TCVDPEDCPV	CEVAGRRFAS	GKKVTLNPSD	PEHCQICHCD

VVNLTCEACQ

- 173 2018203206 08 May 2018

22 01	EPISGTSESA	TPESGPGSEP	ATSGSETPGT	SESATPESGP
GSEPATSGSE 2251	TPGTSESATP	ESGPGTSTEP	SEGSAPGSPA	GSPTSTEEGT
SESATPESGP 2301	GSEPATSGSE	TPGTSESATP	ESGPGSPAGS	PTSTEEGSPA
GSPTSTEEGT 2351	STEPSEGSAP	GTSESATPES	GPGTSESATP	ESGPGTSESA
TPESGPGSEP 2401	ATSGSETPGS	EPATSGSETP	GSPAGSPTST	EEGTSTEPSE
GSAPGTSTEP 2451	SEGSAPGSEP	ATSGSETPGT	SESATPESGP	GTSTEPSEGS
APDSGGGGSG 2501	GGGSGGGGSG	GGGSGGGGSL	VPRGSGGDKT	HTCPPCPAPE
LLGGPSVFLF 2551	PPKPKDTLMI	SRTPEVTCVV	VDVSHEDPEV	KFNWYVDGVE
VHNAKTKPRE 2601	EQYNSTYRVV	SVLTVLHQDW	LNGKEYKCKV	SNKALPAPIE
KTISKAKGQP 2651	REPQVYTLPP	SRDELTKNQV	SLTCLVKGFY	PSDIAVEWES
NGQPENNYKT 2701	TPPVLDSDGS	FFLYSKLTVD	KSRWQQGNVF	SCSVMHEALH
NHYTQKSLSL 2751	SPGK

(b) Cloning of pSYN-FVIII-168, 175, 172 and 174 (Figure 4A-4D) [0352] pSYN-FVIII-168, 172, 174 and 175 are derivatives of pSYN-FVIII-161.

R1645A/R1648A mutations were introduced into pSYN-FVIII-161 to form pSYN-FVIII-168, which produces a SC-FVIII isoform, and an AE288 XTEN was directly fused into the C-terminus of FVIII-HC for further half-life extension. To construct pSYN-FVIII-175, the D'D3 codon sequence was remove form pSYNFVIII-168 for evaluation of the effect of Fc and XTEN technology on FVIII halflife extension.

[0353] To construct pSYN-FVIII-172, the AE288 XTEN fragment was directly fused into the C-terminus of FVIII-HC for further half-life extension, and the D’D3 codon sequence was removed from pSYN-FVIII-172 to form pSYN-FVIII174 for evaluation of the effect of Fc and XTEN technology on FVIII half-life extension.

(c) Cloning of pSYN-FVIII-170 (Figure 4E) [0354] pSYN-FVIII-170 was constructed to evaluate the effect of XTEN and

D’D3 fragment on FVIII half-life extension. The codon sequence VWFD1D2D’D3 fragment and BDD-FVIII were introduced into the 5’ and 3’ end of expression casket, an AE288 XTEN codon sequence which followed by a 35 aa

- 174thrombin cleavable linker was used to connect the VWF and FVIII molecule.

After intra cellular processing, the secreted protein comprises a polypeptide contains the D'D3 fragment of mature VWF molecule which is linked to the N terminus of mature BDD-FVIII by an AE288 XTEN/35 aa thrombin cleavable linker.

pSYN-FVIII-170 protein sequence (SEQ ID NO: 102)

2018203206 08 May 2018

1 SLSCRPPMVK	LVCPADNLRA	EGLECTKTCQ	NYDLECMSMG
CVSGCLCPPG 51 MVRHENRCVA	LERCPCFHQG	KEYAPGETVK	IGCNTCVCRD
RKWNCTDHVC 101 DATCSTIGMA	HYLTFDGLKY	LFPGECQYVL	VQDYCGSNPG
TFRILVGNKG 151 CSHPSVKCKK	RVTILVEGGE	IELFDGEVNV	KRPMKDETHF
EVVESGRYII 201 LLLGKALSVV	WDRHLSISVV	LKQTYQEKVC	GLCGNFDGIQ
NNDLTSSNLQ 251 VEEDPVDFGN	SWKVSSQCAD	TRKVPLDSSP	ATCHNNIMKQ
TMVDSSCRIL 301 TSDVFQDCNK	LVDPEPYLDV	CIYDTCSCES	IGDCAAFCDT
IAAYAHVCAQ 351 HGKVVTWRTA	TLCPQSCEER	NLRENGYEAE	WRYNSCAPAC
QVTCQHPEPL 401 ACPVQCVEGC	HAHCPPGKIL	DELLQTCVDP	EDCPVCEVAG
RRFASGKKVT 451 LNPSDPEHCQ	ICHCDVVNLT	CEACQEPISG	TSESATPESG
PGSEPATSGS 501 ETPGTSESAT	PESGPGSEPA	TSGSETPGTS	ESATPESGPG
TSTEPSEGSA 551 PGSPAGSPTS	TEEGTSESAT	PESGPGSEPA	TSGSETPGTS
ESATPESGPG 601 SPAGSPTSTE	EGSPAGSPTS	TEEGTSTEPS	EGSAPGTSES
ATPESGPGTS 651 ESATPESGPG	TSESATPESG	PGSEPATSGS	ETPGSEPATS
GSETPGSPAG 701 SPTSTEEGTS	TEPSEGSAPG	TSTEPSEGSA	PGSEPATSGS
ETPGTSESAT 751 PESGPGTSTE	PSEGSAPDSG	GGGSGGGGSG	GGGSGGGGSG
GGGSLVPRGS 8 01 GGASATRRYY	LGAVELSWDY	MQSDLGELPV	DARFPPRVPK
SFPFNTSVVY 851 KKTLFVEFTD	HLFNIAKPRP	PWMGLLGPTI	QAEVYDTVVI
TLKNMASHPV 901 SLHAVGVSYW	KASEGAEYDD	QTSQREKEDD	KVFPGGSHTY
VWQVLKENGP 951 MASDPLCLTY	SYLSHVDLVK	DLNSGLIGAL	LVCREGSLAK
EKTQTLHKFI

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1001 LLFAVFDEGK YVNRSLPGLI	SWHSETKNSL	MQDRDAASAR	AWPKMHTVNG
1051 GCHRKSVYWH	VIGMGTTPEV	HSIFLEGHTF	LVRNHRQASL
EISPITFLTA 1101 QTLLMDLGQF	LLFCHISSHQ	HDGMEAYVKV	DSCPEEPQLR
MKNNEEAEDY 1151 DDDLTDSEMD	VVRFDDDNSP	SFIQIRSVAK	KHPKTWVHYI
AAEEEDWDYA 1201 PLVLAPDDRS	YKSQYLNNGP	QRIGRKYKKV	RFMAYTDETF
KTREAIQHES 1251 GILGPLLYGE	VGDTLLIIFK	NQASRPYNIY	PHGITDVRPL
YSRRLPKGVK 1301 HLKDFPILPG	EIFKYKWTVT	VEDGPTKSDP	RCLTRYYSSF
VNMERDLASG 1351 LIGPLLICYK	ESVDQRGNQI	MSDKRNVILF	SVFDENRSWY
LTENIQRFLP 1401 NPAGVQLEDP	EFQASNIMHS	INGYVFDSLQ	LSVCLHEVAY
WYILSIGAQT 1451 DFLSVFFSGY	TFKHKMVYED	TLTLFPFSGE	TVFMSMENPG
LWILGCHNSD 1501 FRNRGMTALL	KVSSCDKNTG	DYYEDSYEDI	SAYLLSKNNA
IEPRSFSQNP 1551 PVLKRHQREI	TRTTLQSDQE	EIDYDDTISV	EMKKEDFDIY
DEDENQSPRS 1601 FQKKTRHYFI	AAVERLWDYG	MSSSPHVLRN	RAQSGSVPQF
KKVVFQEFTD 1651 GSFTQPLYRG	ELNEHLGLLG	PYIRAEVEDN	IMVTFRNQAS
RPYSFYSSLI 1701 SYEEDQRQGA	EPRKNFVKPN	ETKTYFWKVQ	HHMAPTKDEF
DCKAWAYFSD 1751 VDLEKDVHSG	LIGPLLVCHT	NTLNPAHGRQ	VTVQEFALFF
TIFDETKSWY 18 01 FTENMERNCR	APCNIQMEDP	TFKENYRFHA	INGYIMDTLP
GLVMAQDQRI 1851 RWYLLSMGSN	ENIHSIHFSG	HVFTVRKKEE	YKMALYNLYP
GVFETVEMLP 1901 SKAGIWRVEC	LIGEHLHAGM	STLFLVYSNK	CQTPLGMASG
HIRDFQITAS 1951 GQYGQWAPKL	ARLHYSGSIN	AWSTKEPFSW	IKVDLLAPMI
IHGIKTQGAR 2001 QKFSSLYISQ	FIIMYSLDGK	KWQTYRGNST	GTLMVFFGNV
DSSGIKHNIF 2051 NPPIIARYIR	LHPTHYSIRS	TLRMELMGCD	LNSCSMPLGM
ESKAISDAQI 2101 TASSYFTNMF	ATWSPSKARL	HLQGRSNAWR	PQVNNPKEWL
QVDFQKTMKV 2151 TGVTTQGVKS	LLTSMYVKEF	LISSSQDGHQ	WTLFFQNGKV
KVFQGNQDSF 2201 TPVVNSLDPP	LLTRYLRIHP	QSWVHQIALR	MEVLGCEAQD LY

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Example 4: Hydrodynamic injection of XTEN containing FVIIIF/VWF constructs in FVIII and VWF deficient mice [0355] The XTEN containing DNA constructs in Figures 3 and 4 have combined

2-3 half-life extension elements together. To evaluate their FVIII half-life extension potential, a selective group of DNA constructs in figure 3 and figure 4 were introduced into FVIII/VWF double knockout (DKO) mice by Hydrodynamic injection (HDI) at lOOug/mouse dose. Blood samples were then collected by retro orbital blood collection at 24hr post HDI. The post HDI plasma FVIII activity was analyzed by FVIII chromogenic assay, and results were listed in Table 17 and Figure 5. Compared to wild type BDD-FVIII, all XTEN containing DNA constructs yield significantly higher FVIII plasma activity at 24hr post HDI, indicating the corresponding molecules had significant longer circulating protein half-life than BDD-FVIII. The application of the combination of those half-life extending elements was further evaluated in Hemophilic animals.

TABLE 17: FVIII plasma activity 24hr post HDI in FVIII/VWF DKO mice

DNA Construct	BDD- FVIII	FVIII-161	FVIII- 168	FVIII- 172	BDD- FVIII	FVIII- 170
DNA Dose (pg/mouse)	100	100	100	100	50	50
FVIII Activity (mU/mL)	219±72	2446±1012	2209±609	167H223	197±21	399±30

Hydrodynamic injection:

[0356] Hydrodynamic Injection is an efficient and safe non-viral gene delivery method to the liver in small animals, such as mice and rats. It was originally described as a rapid injection of a naked plasmid DNA/saline solution free of endotoxin at a tenth volume of the animal's body weight in about 5-7 seconds.

The naked plasmid DNA contains the gene of interest and the liver produced in a tenth volume of the animal's body weight. The targeted protein is produced in the liver from the injected DNA and can be detected within 24 hours post-injection. Plasma samples were then collected to study the therapeutic property of the expressed protein.

[0357] For all the hydrodynamic injections that were performed herein, 2 ml of plasmid DNA in 0.9% sterile saline solution was delivered via intravenous tail vein injection within about 4-7 seconds to mice weighing 20-35 grams. The mice

- 1772018203206 08 May 2018 [0358] were closely monitored for the first couple of hours until the normal activity resumed. After the blood samples were collected via retro orbital blood collection, plasma samples were then obtained and stored at -80 °C for further analysis.

Example 5: Plasmid construction of co-transfection system for FVIIIFc-VWF Heterodimer contain XTEN insertions (Figure 6)

To increase the protein production yield, two co-transfection systems were generated for protein production, which contains three DNA constructs. The first DNA construct encoded a FVIII-Fc fusion protein in which a AE288 XTEN fragment was directly fuse to the C-terminus of the FVIII heavy chain and followed by either a wild type FVIII light chain fragment (pSYN-FVIII-173, Figure 6B) or a FVIII light chain fragment with R1645A/R1648A mutations (pSYN-FVIII-169, Figure 6A), the FVIII light chain was then directly fused to a single Fc fragment. The second DNA construct is pSYN-VWF-031 which encoding a D'D3-Fc fusion protein (Example 1). HEK293F cells were transfected with the two plasmid along with a third plasmid (PC5) at 80:15:5 ratio. The synthesized proteins were secreted as FVIII (XTEN) Fc/D'D3Fc heterodimer and D'D3Fc dimer and the FVIII (XTEN) Fc/D'D3Fc heterodimer was separated from the D'D3Fc dimer by protein purification.

pSYN-FVIII-169 mature Protein sequence (SEQ ID NO: 103):

ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSVVYKKTL

FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTVVITLKN MASHPVSLHA

101 VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD

151 PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA

201 VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR

251 KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL

301 MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL

351 TDSEMDVVRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL

401 APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG

451 PLLYGEVGDT LLIIFKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD

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501 FPILPGEIFK RDLASGLIGP	YKWTVTVEDG	PTKSDPRCLT	RYYSSFVNME
551 LLICYKESVD IQRFLPNPAG	QRGNQIMSDK	RNVILFSVFD	ENRSWYLTEN
601 VQLEDPEFQA SIGAQTDFLS	SNIMHSINGY	VFDSLQLSVC	LHEVAYWYIL
651 VFFSGYTFKH GCHNSDFRNR	KMVYEDTLTL	FPFSGETVFM	SMENPGLWIL
701 GMTALLKVSS SFSQNGAPGT	CDKNTGDYYE	DSYEDISAYL	LSKNNAIEPR
751 SESATPESGP SGSETPGTSE	GSEPATSGSE	TPGTSESATP	ESGPGSEPAT
801 SATPESGPGT ESGPGSEPAT	STEPSEGSAP	GSPAGSPTST	EEGTSESATP
851 SGSETPGTSE EEGTSTEPSE	SATPESGPGS	PAGSPTSTEE	GSPAGSPTST
901 GSAPGTSESA GSEPATSGSE	TPESGPGTSE	SATPESGPGT	SESATPESGP
951 TPGSEPATSG STEPSEGSAP	SETPGSPAGS	PTSTEEGTST	EPSEGSAPGT
1001 GSEPATSGSE PVLKRHQAEI	TPGTSESATP	ESGPGTSTEP	SEGSAPASSP
1051 TRTTLQSDQE FQKKTRHYFI	EIDYDDTISV	EMKKEDFDIY	DEDENQSPRS
1101 AAVERLWDYG GSFTQPLYRG	MSSSPHVLRN	RAQSGSVPQF	KKVVFQEFTD
1151 ELNEHLGLLG SYEEDQRQGA	PYIRAEVEDN	IMVTFRNQAS	RPYSFYSSLI
1201 EPRKNFVKPN VDLEKDVHSG	ETKTYFWKVQ	HHMAPTKDEF	DCKAWAYFSD
1251 LIGPLLVCHT FTENMERNCR	NTLNPAHGRQ	VTVQEFALFF	TIFDETKSWY
1301 APCNIQMEDP RWYLLSMGSN	TFKENYRFHA	INGYIMDTLP	GLVMAQDQRI
1351 ENIHSIHFSG SKAGIWRVEC	HVFTVRKKEE	YKMALYNLYP	GVFETVEMLP
1401 LIGEHLHAGM GQYGQWAPKL	STLFLVYSNK	CQTPLGMASG	HIRDFQITAS
1451 ARLHYSGSIN QKFSSLYISQ	AWSTKEPFSW	IKVDLLAPMI	IHGIKTQGAR
1501 FIIMYSLDGK NPPIIARYIR	KWQTYRGNST	GTLMVFFGNV	DSSGIKHNIF
1551 LHPTHYSIRS TASSYFTNMF	TLRMELMGCD	LNSCSMPLGM	ESKAISDAQI
1601 ATWSPSKARL TGVTTQGVKS	HLQGRSNAWR	PQVNNPKEWL	QVDFQKTMKV
1651 LLTSMYVKEF TPVVNSLDPP	LISSSQDGHQ	WTLFFQNGKV	KVFQGNQDSF
1701 LLTRYLRIHP CPAPELLGGP	QSWVHQIALR	MEVLGCEAQD	LYDKTHTCPP

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17	51 SVFLFPPKPK	DTLMISRTPE	VTCVVVDVSH	EDPEVKFNWY
18	VDGVEVHNAK 01 TKPREEQYNS	TYRVVSVLTV	LHQDWLNGKE	YKCKVSNKAL
18	PAPIEKTISK 51 AKGQPREPQV	YTLPPSRDEL	TKNQVSLTCL	VKGFYPSDIA
19	VEWESNGQPE 01 NNYKTTPPVL	DSDGSFFLYS	KLTVDKSRWQ	QGNVFSCSVM
HEALHNHYTQ 1951 KSLSLSPGK pSYN-FVIII-173 mature Protein seuuencing (SEO ID NO: 104):
	1 ATRRYYLGAV	ELSWDYMQSD	LGELPVDARF	PPRVPKSFPF
	NTSVVYKKTL 51 FVEFTDHLFN	IAKPRPPWMG	LLGPTIQAEV	YDTVVITLKN
-	MASHPVSLHA 01 VGVSYWKASE	GAEYDDQTSQ	REKEDDKVFP	GGSHTYVWQV
-i	LKENGPMASD 51 PLCLTYSYLS	HVDLVKDLNS	GLIGALLVCR	EGSLAKEKTQ
/->	TLHKFILLFA 01 VFDEGKSWHS	ETKNSLMQDR	DAASARAWPK	MHTVNGYVNR
/·> z	SLPGLIGCHR 51 KSVYWHVIGM	GTTPEVHSIF	LEGHTFLVRN	HRQASLEISP
3	ITFLTAQTLL 01 MDLGQFLLFC	HISSHQHDGM	EAYVKVDSCP	EEPQLRMKNN
3	EEAEDYDDDL 51 TDSEMDVVRF	DDDNSPSFIQ	IRSVAKKHPK	TWVHYIAAEE
EDWDYAPLVL 401 APDDRSYKSQ	YLNNGPQRIG	RKYKKVRFMA	YTDETFKTRE
4	AIQHESGILG 51 PLLYGEVGDT	LLIIFKNQAS	RPYNIYPHGI	TDVRPLYSRR
5	LPKGVKHLKD 01 FPILPGEIFK	YKWTVTVEDG	PTKSDPRCLT	RYYSSFVNME
5	RDLASGLIGP 51 LLICYKESVD	QRGNQIMSDK	RNVILFSVFD	ENRSWYLTEN
6	IQRFLPNPAG 01 VQLEDPEFQA	SNIMHSINGY	VFDSLQLSVC	LHEVAYWYIL
6	SIGAQTDFLS 51 VFFSGYTFKH	KMVYEDTLTL	FPFSGETVFM	SMENPGLWIL
7	GCHNSDFRNR 01 GMTALLKVSS	CDKNTGDYYE	DSYEDISAYL	LSKNNAIEPR
·?	SFSQNGAPGT 51 SESATPESGP	GSEPATSGSE	TPGTSESATP	ESGPGSEPAT
o <'	SGSETPGTSE 01 SATPESGPGT	STEPSEGSAP	GSPAGSPTST	EEGTSESATP
8	ESGPGSEPAT 51 SGSETPGTSE	SATPESGPGS	PAGSPTSTEE	GSPAGSPTST
9	EEGTSTEPSE 01 GSAPGTSESA	TPESGPGTSE	SATPESGPGT	SESATPESGP
9	GSEPATSGSE 51 TPGSEPATSG	SETPGSPAGS	PTSTEEGTST	EPSEGSAPGT
	STEPSEGSAP

- 180

2018203206 08 May 2018

10	01 GSEPATSGSE PVLKRHQREI	TPGTSESATP	ESGPGTSTEP	SEGSAPASSP
10	51 TRTTLQSDQE FQKKTRHYFI	EIDYDDTISV	EMKKEDFDIY	DEDENQSPRS
11	01 AAVERLWDYG GSFTQPLYRG	MSSSPHVLRN	RAQSGSVPQF	KKVVFQEFTD
11	51 ELNEHLGLLG SYEEDQRQGA	PYIRAEVEDN	IMVTFRNQAS	RPYSFYSSLI
12	01 EPRKNFVKPN VDLEKDVHSG	ETKTYFWKVQ	HHMAPTKDEF	DCKAWAYFSD
12	51 LIGPLLVCHT FTENMERNCR	NTLNPAHGRQ	VTVQEFALFF	TIFDETKSWY
1301 APCNIQMEDP RWYLLSMGSN	TFKENYRFHA	INGYIMDTLP	GLVMAQDQRI
13	51 ENIHSIHFSG SKAGIWRVEC	HVFTVRKKEE	YKMALYNLYP	GVFETVEMLP
14	01 LIGEHLHAGM GQYGQWAPKL	STLFLVYSNK	CQTPLGMASG	HIRDFQITAS
14	51 ARLHYSGSIN QKFSSLYISQ	AWSTKEPFSW	IKVDLLAPMI	IHGIKTQGAR
15	01 FIIMYSLDGK NPPIIARYIR	KWQTYRGNST	GTLMVFFGNV	DSSGIKHNIF
15	51 LHPTHYSIRS TASSYFTNMF	TLRMELMGCD	LNSCSMPLGM	ESKAISDAQI
1601 ATWSPSKARL TGVTTQGVKS	HLQGRSNAWR	PQVNNPKEWL	QVDFQKTMKV
16	51 LLTSMYVKEF TPVVNSLDPP	LISSSQDGHQ	WTLFFQNGKV	KVFQGNQDSF
1701 LLTRYLRIHP CPAPELLGGP	QSWVHQIALR	MEVLGCEAQD	LYDKTHTCPP
17	51 SVFLFPPKPK VDGVEVHNAK	DTLMISRTPE	VTCVVVDVSH	EDPEVKFNWY
18	01 TKPREEQYNS	TYRVVSVLTV	LHQDWLNGKE	YKCKVSNKAL

PAPIEKTISK

1851 AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE

1901 NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ

1951 KSLSLSPGK

Example 6. Protein purification for FVIII- 169/VWF-031 and FVIII-173/VWF 031 [0359] A tangential flow filtration (TFF) step was used to buffer exchange the clarified conditioned media. The FVIII-169/VWF-031 or FVIII-173/VWF-031 heterodimer was then purified using a two-step chromatography process. A weak anion exchange resin was used, followed by affinity chromatography. The final purified product had acceptable purity by SEC-HPLC. The specific activity was

- 181 2018203206 08 May 2018 [0360] [0361] compatible to B-domain deleted FVIII, as measured by FVIII chromogenic assay and A280 concentration. Purity and the presence of each moiety of this molecule were confirmed by SDS-PAGE and western blotting.

Example 7. Evaluation the VWF binding ability of FVIII- 169/VWF-031 by Octet assay

The VWF binding ability of FVIII- 169/VWF-031 was obtained by BioLayer Interferometry (BLI) based measurements (Octet assay) at 25 °C with a ForteBio Octet 384 instrument, using Tris binding buffer (50 mM Tris, pH 7.2,

150 mM NaCI, 5 mM CaCF). The Octet assay for determining FVIII binding was based on the hydrophobic immobilization of Human von Willebrand Factor (Haematologic Technologies Catalog No. HCVWF-0191) onto the APS Biosensor, then followed by the binding of 1.0% Bovine Serum Albumin (Jackson ImmunoResearch Catalog No. 001-000-161). Briefly, hvWF (20 pg/mL) was diluted in Tris buffer and loaded across APS Biosensors for 600 sec, yielding approximately 3.0-3.5 nm binding on the reaction probes. Control APS probes were loaded with 1.0% BSA in the absence of hvWF for reference subtraction. After loading, all probes were incubated in Tris buffer for 300 sec to establish a new baseline. Subsequently, biosensor probes were incubated in solutions of FVIII-XTEN 169 or FVIIIFc Drug Substance (0, 0.6, 2, 6, 20, 60, 200, 600 IU/mL) for 5 min at room temperature, followed by a 5 min dissociation step. Using the Octet data analysis software, the binding response (nm) was derived from the subtracted data (Reaction probe minus Reference probe). No binding to immobilized VWF was detected for FVIII-169/VWF-031 (Figure 7), indicating a complete shielding of FVIII from full length VWF molecule by the D'D3 fragment.

Example 8. FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO mice

The PK profile of FVIII-169/VWF-031 was tested in HemA and FVIII/VWF DKO mice to evaluate the ability of the D'D3 fragment to shield the FVIII moiety from the endogenous VWF. HemA or FVIII/VWF DKO mice were treated with a single intravenous dose of FVIII- 169/VWF-031 at 200 IU/kg, plasma samples were then collected at 5min, 8hr, 24hr, 48hr and 72 hours post dosing. The FVIII activity of plasma sample was tested by FVIII chromogenic

- 1822018203206 08 May 2018 assay, and half-life of FVIII- 169/VWF-031 was calculated using WinNonlin program.

[0362] Complete inhibition of the constructs' binding to immobilized VWF was demonstrated by biolayer interferometry (Figure 7) for FVIII- 169/VWF-031. This indicates the D'D3 fragment in the molecule had successfully blocked the FVIII binding to native VWF molecules, therefor similar half-life of FVIII-169/VWF031 was predicted in the two different mouse strains. As shown in Figure 8A and Table 18, as expected, FVIII-169/VWF-031 had similar PK profile in both HemA and FVIII/VWF DKO mice, which has demonstrated that the half-life of FVIIIFc/VWF heterodimer is independent from the half-life of endogenous VWF. The separation of the FVIIIFc/VWF heterodimer half-life from the endogenous VWF half-life , eliminated the FVIII extension ceiling and opened the possibility of further extending FVIII half-life beyond the 2 fold half-life limit imposed by endogenous VWF.

TABLE 18: FVIII-169/VWF-031 PK in HemA and FVIII/VWF DKO mice

Mouse Strain	Recover y (%)	tl/2 (hr)	MRT (hr)	Cl (mL/hr/k g)	Vss (mL/kg )	AUC (hr*kg*mIU/mL /mlU)
FVIII/VW FDKO	69	17.94	20.1	4.06	81.69	0.2461
HemA	83	16.65	18.44	3.57	85.72	0.28

[0363] The FVIII protecting ability of the XTEN insertion and D'D3 fragment was evaluated by comparing the half-life of FVIII- 169/VWF-031 with FVIII-169/Fc and FVIIIFc in FVIII/VWF DKO mice. After a single IV administration, blood samples were collected at 5min, 8hr, 24hr, 48hr and 72hr for FVIII-169/VWF031, 5min, 8hr, 24hr, 32hr, 48hr for FVIII-169/Fc and at 5min, 1, 2, 4, 6 and 8hrs for FVIIIFc. The FVIII activity of plasma sample was tested by FVIII chromogenic assay, and half-life of FVIII-155/VWF-031 was calculated using WinNonlin program.

[0364] The study results were summarized in Figure 8B and Table 19, rFVIIIFc has a 1,6hr half-life in DKO mice due to the loss of VWF protection. When an XTEN insertion was introduced into the FVIIIFc molecule, the resulting FVIII - 183 2018203206 08 May 2018

169/Fc molecule has a 7hr half-life, a 4 fold half-life extension by the XTEN insertion. Finally, when D'D3 fragment was incorporated into the molecule to form FVIII- 169/VWF-031, a 17hr half-life was observed, another 2.5 fold further increase by the D'D3 fragment. In addition of the half-life improvement, improved Mean residency time (MRT), Clearance (Cl) and AUC were also observed as shown in Table 19.

[0365] FVIII-169/VWF-031 has achieved 17-18hr ti/₂ in both HemA and

FVIII/VWF DKO mice, which is the upper limit of the 1i/₂ extension ceiling that imposed by VWF clearance. More ti/₂ extension elements can be further incorporated into this molecule, such as a second XTEN insertion within FVIII. The synergistic effect of D'D3 fragment and XTEN insertions provided the possibility of the complete protection for FVIII from its clearance pathway, a final breakthrough of the 2 fold FVIII 11/₂ extension limit might be achieved by the FVIIIFc/XTEN/VWF variants.

TABLE 19. FVIII-169/VWF-031 PK in FVIII/VWF DKO mice

Mouse Strain	Treatment	Recover y (%)	tl/2 (hr)	MRT (hr)	Cl (mL/hr/k g)	Vss (mL/kg )	AUC/D (hr*kg* mlU/mL /mlU)
FVIII/VW FDKO	rFVIIIFc	35	1.6	2.1	57.7	120.2	0.0173
rFVIII- 169/ Fc	77	7.0	6.2	6.4	39.2	0.1573
rFVIII- 169/ VWF-031	69	17.9	20.1	4.1	81.7	0.2461

Example 9: FVIII-XTEN variants cell media concentrate PK in D'D3 expressing FVIII/VWF DKO mice [0366] The ability of D'D3 fragment to extend the ti/₂ of FVII-XTEN was evaluated in the D'D3 expressing FVIII/VWF DKO mouse model (described in example 2). In this study, instead of using VWF-025 to introduce the DD3 dimer into the circulation, VWF-029 construct was used to introduce the DD3 monomer into the circulation. To prepare FVIII-XTEN variants protein, a small scale (5 ΟΙ 00 mL) transient transfection culture media was prepared, at day 4 post transfection, cell culture was harvested and concentrated to reach 10-20 IU/mL of

- 1842018203206 08 May 2018

FVIII activity range which is suitable for PK study. The concentrated cell media were then used for standard PK study in FVIII/VWF DKO mice with or without

D'D3 in the circulation.

[0367] Total of 6 FVIII-ΧΤΕΝ variants that contains 1-3 XTEN insertions were tested in the system, their ti/2 were summarized in Table 20 and data from representative variants were plotted in Figure 9A.

[0368] Longer half-life was observed for all the FVIII-ΧΤΕΝ variants with the presents of DD3 fragment in the circulation (Table 20), which demonstrated the D'D3 protection for FVIII-ΧΤΕΝ from its clearance pathways. Furthermore, when compared to its 14hr half-life in HemA mice, LSD0055.021 has a 20.4hr ti/2 in DD3 expressing DKO mice (Figure 9B, Table 20), indicates the final breakthrough of the 2 fold half-life extension ceiling for FVIII molecules. By further modify the FVIII(XTEN)/VWF molecule, we could potentially achieve even longer FVIII ti/2, and provide HemA patients a FVIII protein that only requires once weekly or less frequent dosing regimen.

TABLE 20. FVIII-XTEN ti_/2 in D'D3 expressing FVIII/VWF DKO mice

FVIII-XTEN ID	# of XTEN insertions	Insertion sites	XTEN size	ti/2 (hr) DKO mice	ti/2 (hr) pLIVEDD3/ DKO mice	tl/2 (hr) HemA mice
pSD-0013	1	CT	144	3.3	7.9
LSD0003.009	2	B*/CT	144/288	9.7	16.4
LSD0038.015	2	1656/26	144/144	7.8	17.2
LSD0049.002	3	18/B*/CT	144/144/288	12.6	17.5
LSD0051.002	3	403/B*/CT	144/144/288	11.1	19.9
LSD0055.021	3	1900/B*/CT	144/144/288	16	20.4	14

*B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

Example 10: Stability of VWF- and XTEN-containing FVIII variants in FVIII/VWF double knockout (DKO) plasma [0369] Plasma stability of rFVIIIFc protein variants was tested in FVIII/VWF double knockout (DKO) mouse plasma. For the stability assay, HEK293 cells were co-transfected with plasmids directing the expression of rFVIIIFc or FVIII 169 (rFVIIIFc with 288 AE XTEN inserted at the B-domain junction) and plasmids directing the expression of either IgG-Fc or VWF-031 (VWF D'D3 region fused to IgG-Fc). At day four post-transfection, cell culture media was

- 185 2018203206 08 May 2018 [0370] [0371] [0372] harvested and concentrated to 30 IU/mL based on FVIII chromogenic activity. Concentrated cell culture medium was then added into DKO mouse plasma to yield a FVIII activity of 5 IU/mL and incubated at 37°C. Aliquots were collected at different time points for activity measurement by chromogenic assay. Activity at each time point was measured in duplicate, and the average activity was plotted as a function of time. The activity of FVIIIFc, a dual chain (de) FVIII molecule in which heavy and light chains are held together by non-covalent interaction, decreases with time in DKO mouse plasma (Figure 10). The activity of FVIII169:Fc, which contains a 288 AE XTEN insertion at the B-domain junction, decays at a reduced rate relative to rFVIIIFc, indicating that enhanced stability is conferred by the XTEN insertion. Given that VWF has been proposed to enhance the stability of FVIII in vivo, we evaluated the plasma stability ofFVIII169:VWF-031. This heterodimeric molecule, in which the FVIII element and the VWF D'D3 element are fused to respective hemi-domains of Fc, exhibited additional plasma stability relative to FVIII-169:Fc, indicating that the VWF DD3 domain and XTEN have a synergistic effect on the plasma stability of rFVIIIFc. Example 11: The effect on FVIII half-life of Fc fusion, XTEN insertion and the D'D3 fragment of VWF.

To assess the effect of Fc fusion, XTEN insertion and D'D3 fragment of VWF on the half-life of FVIII, the pharmacokinetic properties of B domain deleted recombinant FVIII (rBDD-FVIII), rFVIIIFc, FVIII-169:Fc and FVIII169:VWF-031 were evaluated in FVIII/VWF double knockout (DKO) mice.

DKO mice were treated with a single intra venous administration of 200 IU/kg of FVIII proteins, and plasma samples were collected at designated time points as indicated in Figure 11. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay and half-life was calculated using the WinNonlin-Phoenix program. The pharmacokinetic parameters of the tested molecules are listed in Table 21. The time regression curve of plasma FVIII activity for each FVIII variants were plotted in Figure 11.

Unmodified BDD-FVIII had a half-life of 0.23 hr in DKO mice, the FVIIIFc fusion protein has an extended half-life of 1.66 hr in DKO mice due to the recycling of FVIIIFc protein through the Fc:FcRn interaction. When a 288 residue of AEXTEN polypeptide was incorporated into the B domain region of

- 1862018203206 08 May 2018

FVIII within the FVIIIFc molecule, the half-life of the resulting FVIII169/Fc protein was further extended to 7.41 hr in DKO mice. Finally, with the addition of the D'D3 domain of VWF, the half-life of FVIII 169/VWF031 heterodimer has reached 17.9 hr in DKO mice (Figure 11, Table 21). In addition of the half-life, all of the other PK parameters also improved proportionally with the addition of each element (Table 21). FVIII can tolerate multiple half-life extension elements, and this synergistic effect of the three elements on FVIII half-life extension, enabled the further improvement of the half-life of FVIII-ΧΤΕΝ VWF heterodimers.

Table 21: PK parameters of FVIII variants

FVIII	FVIII Isofor m	XTEN	Insertions	T 1/2 (hr)	MRT (hr)	Cl (mL/hr /kg)	Vss (mL/kg )	AUC D kg*hr/ mL
Site	XTEN Length
BDD-FVIII	dc			0.23	0.24	407.72	97.42	0.0025
FVIIIFc	dc			1.66	2.06	62.66	128.82	0.0161
FVIII 169/Fc	sc	B*	AE288	7.41	6.67	6.24	41.61	0.1603
FVIII 169/VWFO 31	sc	B*	AE288	17.94	20.1	4.06	81.69	0.2463

*B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

Example 12: Pharmacokinetic properties of different FVIII-XTENVWF heterodimers [0373] To evaluate the combined effect of the VWF-D'D3 fragment and XTEN insertions on the FVIII half-life, the pharmacokinetic properties of FVIII-XTENFc:VWF-Fc heterodimers were tested in HemA mice and compared to those of the single chain isoform of BDD-FVIII (scBDD-FVIII) and FVIII-169:VWF-031 (example 10). Seven new FVIII-ΧΤΕΝ-Fc constructs were generated (protein sequences were listed in Table 24). Schematic diagrams of those constructs are shown in Figure 14A-H. FVIII-195 and FVIII-199, respectively, are the FVIII dual chain and single chain isoforms that each contains two XTEN insertions at positions 1900 and 1656. FVIII-196 and FVIII-201, respectively, are the FVIII dual chain and single chain isoforms that each contains three XTEN insertions at positions 26, 1656 and 1900. FVIII-203, -204 and -205 are sc-FVIIIFc molecules with two XTEN insertions at the B domain junction and at positions 1900, 403 or 18, respectively. Each FVIII-ΧΤΕΝ-Fc construct was co-expressed with VWF- 1872018203206 08 May 2018 [0374]

031 in HEK293 cells to produce FVIII-XTEN-Fc/VWF heterodimeric proteins.

At day four post-transfection, cell culture medium was harvested and either concentrated to 20 IU/mL based on FVIII chromogenic activity (FVIII-195: VWF031, FVIII-196 :VWF-031, FVIII-199 :VWF-031, FVIII-203:VWF-031 and FVIII204: VWF-031) or purified (scBDD-FVIII, FVIII-169:VWF-031, FVIII201:VWF-031 and FVIII-205:VWF-031). Having demonstrated the complete intra-molecular shielding of FVIII molecule from the endogenous VWF by the D'D3 fragment in the FVIII-XTEN-Fc:VWF-Fc heterodimer (FVIII-169:VWF031, Example 5), HemA mice was chosen for the PK evaluations. Purified protein or concentrated cell culture medium was administered to 8-12 week-old HemA mice by intravenous administration at a dose of 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 8 hr, 16 hr, 24 hr, 32 hr, 48 hr, 72 hr and 96 hr post-dosing. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay and half-life was calculated using the WinNonlin-Phoenix program. The pharmacokinetic parameters of the tested molecules are listed in Table 22. The plasma FVIII activities at selected time points for FVIII-XTENFc/VWF-Fc variants were plotted in Figures 12A-C.

When XTEN was inserted into positions 1900 and 1656 (FVIII-195,

FVIII-199), moderate improvement in half-life was observed for the scFVIII isoform (FVIII-199:VWF-031) compared to FVIII-169:VWF-031. However, the dcFVIII iso form exhibited a shorter half-life than did FVIII-169: VWF-031, indicating that the single chain isoform might be significantly more stable than the corresponding dual chain isoform (Table 22 and Figure 12A). When a third XTEN insertion was incorporated into FVIII-199 at position 26, the half-life of the resulting molecule FVIII-201:VWF-031 had reached 24.6 hr, which represents greater than a threefold half-life improvement relative to scBDD-FVIII (Table 22 and Figure 12C). We have also tested the half-life extension effect of the second XTEN insertion at position 403 (A2 domain), 1900 (A3 domain) and 18 (A 1 domain) each in combination with the B domain XTEN insertion. While the addition of the A2 or A3 XTEN insertion did not confer an additional half-life benefit (Table 22, Figure 12b), the addition of the Al insertion further extended the half-life of the FVIII-XTEN-Fc:VWF-Fc heterodimer to 29.4 hr (Table 22, Figure 12C), which is greater than threefold longer than that of scBDD-FVIII.

- 188 2018203206 08 May 2018 [0375] When XTENs were incorporated into the FVIIIFc/VWF heterodimer construct, degree of half-life improvement of the resulting molecules was variable, and no obvious correlation was observed between half-lives and either the site or number of XTEN insertion, suggesting that the half-life of the FVIII-XTENFc/VWF heterodimer is determined by the integrity of the whole molecule rather than by the number or placement of XTEN insertions.

[0376] The 24.6 hr and 29.4 hr half-lives observed for FVIII-XTEN-Fc:VWF-Fc heterodimers clearly exceeded the 1.6- to 2-fold limitation on FVIII half-life extension. If this finding translates for HemA patients, it will allow once-weekly or less frequent dosing for FVIII prophylaxis.

Table 22: PK parameters of FVIII-XTEN-Fc/VWF-Fc heterodimers

FVIII	FVIII Iso for m	XTEN Insertions	T 1/2 (hr)	MRT (hr)	Cl (mL/h r/kg)	Vss (mL/k g)	AUC D kg*hr/ mL
Site	XTEN Length
scBDD-FVIII	sc			7.16	10.1 6	4.38	44.44	0.23
FVIII 169/VWFO 31	sc	B*	AE288	16.6 5	18.4 4	3.57	65.79	0.28
FVIII 195/VWFO	dc	1656/190	AG 144/	12.5	13.8	9.04	125.4	0.11
31	0	AE144	6	8	8
FVIII 199/VWFO	sc	1656/190	AG 144/	18.5	20.0	6.24	125.2	0.16
31	0	AE144	7	9	8
FVIII201/VWF0 31	sc	26/1656/ 1900	AG144/AG144/AE1 44	24.6 3	33.6 7	1.9	63.97	0.53
FVIII203/VWF0 31	sc	403/B*	AE144/AE288	15.5 2	18	3.65	65.61	0.27
FVIII204/VWF0 31	sc	1900/B*	AE144/AE288	16.3	20.6 3	2.87	59.14	0.35
FVIII205/VWF0 31	sc	18/B*	AE144/AE288	29.4	37.0 6	1.82	67.39	0.55

*B indicates an XTEN sequence (e.g., 144) is inserted immediately downstream of amino acid residue 745 corresponding to mature FVIII sequence.

[0377] In addition to incorporating XTEN into the FVIII molecule, we also evaluated the potential half-life extension benefit of incorporating XTEN as a linker between the D'D3 and Fc fragment. FVIII-155 (scFVIIIFc) was coexpressed with VWF-034 (VWF-Fc with AE 288 XTEN plus a 35 residue thrombin cleavable linker) in HEK293 cells. At day 4 post-transfection, cell culture medium was harvested and concentrated to 20 IU/mL based on FVIII activity assay. FVIII/VWF DKO mice were dosed with concentrated cell culture

- 1892018203206 08 May 2018 [0378] media at 200 IU/10 mL/kg with a single intravenous injection. Plasma samples were collected at 5 min, 8 hr, 24 hr, 48 hr, 72 hr and 96 hr post-dosing. The FVIII activity of plasma samples was analyzed by FVIII chromogenic assay, and the regression curve of plasma FVIII activity as a function of time was plotted (Figure 13). FVIII-155/VWF-034 exhibited the same improvement in half-life as FVIII169/VWF-031, which has AE 288 XTEN inserted into the B domain junction of FVIII, as illustrated by the over lapping regression curves for the two molecules (Figure 13). The demonstration that XTEN insertion into the VWF-Fc polypeptide confers half-life improvement of a magnitude similar to that conferred by XTEN insertion at the B domain junction of the FVIII polypeptide suggests that further half-life improvement may be possible in a heterodimeric molecule in which intra-molecular XTEN insertion in the FVIII polypeptide is combined with inter-domain XTEN insertion between the VWF and Fc elements of the VWF-Fc polypeptide.

Example 13A: Pharmacokinetic properties of additional FVIII-XTENVWF heterodimers

In addition to the FVIII-XTEN VWF heterodimers that were listed in

Table 22, FVIII-XTEN VWF heterodimers containing different composition of

XTEN insertions, single chain and dual chain version of FVIII (Table 23 A) are either tested or will be tested in HemA for their pharmacokinetic properties.

Various FVIII constructs (Table 23B) and VWF constructs (Table 23C) are also disclosed below. HemA mice will be treated with a single dose of intravenous administration of the heterodimer proteins at 200 IU/10 mL/kg. Plasma samples will then be collected at 5 min, 24, 48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma samples will be analyzed by FVIII chromogenic assay and half-life will be calculated using the WinNonlin-Phoenix program. The protein sequences of the listed heterodimers were listed in Table 25.

Table 23A. Plausible FVIII-XTEN-Fc:VWF-Fc heterodimer combinations for PK and activity improvement.

	pSYN VWF015	pSYN VWF031	pSYN VWF034 **	pSYN VWF-036
pSYN FVIII 010	-	ti/2 8.7 hr DKO mice	To be tested	-
pSYN FVIII 155	ti/2 6.3 hr DKO mice	ti/2 10.8 hr HemA mice	ti/218.6 hr HemA mice	ti/2 13.3 hr HemA mice

- 1902018203206 08 May 2018

pSYN FVIII 169 **	-	ti/2 16.7 hr Hem A mice	ti/2 31.1 hr Hem A mice	-
pSYN FVIII 173 **	-	ti/2 15.2 hr DKO mice	ti/2 28.9 hr Hem A mice	To be tested
pSYN FVIII 205		ti/2 29.4 hr Hem A mice	ti/2 32.4 hr Hem A mice	ti/2 29.7 hr Hem A mice
pSYN FVIII 266		ti/2 24.5 hr Hem A mice	ti/2 27.4 hr Hem A mice	-
pSYN FVII 267	-	ti/2 23.0 hr Hem A mice	ti/2 25.7 hr Hem A mice
pSYN FVIII 268		To be tested	To be tested	To be tested
Dual chain isoform of pSYN FVIII 268		To be tested	To be tested	To be tested
**Fength of Xr	ΈΝ can be changed to 72, 144, 288, 324, 333, 576, or 864.

Table 23B. FVIII Constructs:

pSYN FVIII 010	dual chain FVIIIFc
pSYN FVIII 169	Single chain FVIIIFc with 288 AE XTEN in B-domain
pSYN FVIII 173	dual chain FVIIIFc with 288 AE XTEN in B-domain
pSYN FVIII 195	dual chain FVIIIFc with two 144 XTENs at amino acid 1656 and 1900
pSYN FVIII 196	dual chain FVIIIFc with three 144 XTENs at amino acid 26, 1656 and 1900
pSYN FVIII 199	Single chain FVIIIFc with two 144 XTENs at amino acid 1656 and 1900
pSYN FVIII 201	Single chain FVIIIFc with three 144 XTENs at amino acid 26, 1656 and 1900
pSYN FVIII 203	Single chain FVIIIFc with 144 AE XTEN at amino acid 1900 and 288 AE XTEN in B-domain
pSYN FVIII 204	Single chain FVIIIFc with 144 AE XTEN at amino acid 403 and 288 AE XTEN in B-domain
pSYN FVIII 205	Single chain FVIIIFc with 144 AE XTEN at amino acid 18 and 288 AE XTEN in B-domain
pSYN FVIII 207	Single chain FVIII ( no Fc, no XTEN)
pSYN FVIII 266	Single chain FVIIIFc with 42 AE XTEN at amino acid 18 and 288 AE XTEN in B-domain
pSYN FVIII 267	Single chain FVIIIFc with 72 AE XTEN at amino acid 18 and 288 AE XTEN in B-domain
pSYN FVIII 268	Single chain FVIIIFc with 144 AE XTEN at amino acid 18
pSYN FVIII	Single chain FVIIIFc with 72 AE XTEN at amino acid 18

- 191 2018203206 08 May 2018

269
pSYN FVIII 271	Single chain FVIIIFc with 42 AE XTEN at amino acid 18
pSYN FVIII 272	Single chain FVIII with 144 AE XTEN at amino acid 18 and 288 AE XTEN in B-domain (no Fc)

Table 23C. VWF Constructs:

pSYN VWF031	VWF-D1D2D'D3- 48aa long thrombin cleavable GS linker-Fc with C1099A/C1142 A
pSYN VWF034	VWF-D1D2D'D3- 288AE XTEN +35aa long thrombin cleavable GS linker-Fc with C1099A/C1142A
pSYN VWF035	VWF-D1D2D'D3- 72aa long thrombin cleavable GS linker-Fc with C1099A/C1142 A
pSYN VWF036	VWF-D1D2D'D3- 98aa long thrombin cleavable GS linker-Fc with C1099A/C1142 A
pSYN VWF041	VWF-D1D2DO3 with 288 AE XTEN in D3 and 48aa long thrombin cleavable GS linker after D3-Fc with C1099A/C1142A

Example 13B: Pharmacokinetic properties of additional FVIII-XTENVWF heterodimers.

[0379] FVIII-XTEN VWF heterodimers were tested in HemA mice for their pharmacokinetic properties. The heterodimers tested are FVIII 169/VWF034, FVIII205/VWF034, FVIII205/VWF036 and FVIII266/VWF031. HemA mice were administered with a single intravenous dose of various heterodimer proteins at 200 IU/10 mL/kg. Plasma samples were collected at 5 min, 24, 48, 72, 96 and 120 hrs post-dosing. FVIII activity of the plasma samples were analyzed by FVIII chromogenic assay, and half-lives were calculated using the WinNonlin-Phoenix program. The PK results are shown below in Table 24.

Table 24. Additional FVIII-XTEN_VWF - PK in HemA Mice

Treatment	5min recovery (%)	HL (hr)	MRT (hr)	Cl (mL/hr/ kg)	Vss (mL/kg)	AUCD (hr*kg*m IU/ mL/mlU)	Fold of ti/2 increase vs scBDD- FVIII
ScBDD- FVIII		7.16	10.16	4.83	44.44	0.23	-
FVIII 169/V WF034	76	31.1	34.57	1.73	59.77	0.58	4.3
FVIII205/V WF034	68	32.41	39.79	1.55	61.73	0.64	4.6
FVIII205/V WF036	74	29.71	36.35	1.61	58.43	0.62	4.1
FVIII266/V	66	24.45	22.75	2.67	60.83	0.37	3.4

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WF031

pSYNFVIII 010 nucleotide sequence-(Dual chain FVIIIFc) (SEQ ID NO:

1 ATGCAAATAG TGCGATTCTG	AGCTCTCCAC	CTGCTTCTTT	CTGTGCCTTT
51 CTTTAGTGCC CTGTCATGGG	ACCAGAAGAT	ACTACCTGGG	TGCAGTGGAA
101 ACTATATGCA AAGATTTCCT	AAGTGATCTC	GGTGAGCTGC	CTGTGGACGC
151 CCTAGAGTGC TGTACAAAAA	CAAAATCTTT	TCCATTCAAC	ACCTCAGTCG
201 GACTCTGTTT GCTAAGCCAA	GTAGAATTCA	CGGATCACCT	TTTCAACATC
251 GGCCACCCTG TGAGGTTTAT	GATGGGTCTG	CTAGGTCCTA	CCATCCAGGC
301 GATACAGTGG CTGTCAGTCT	TCATTACACT	TAAGAACATG	GCTTCCCATC
351 TCATGCTGTT GCTGAATATG	GGTGTATCCT	ACTGGAAAGC	TTCTGAGGGA
401 ATGATCAGAC CTTCCCTGGT	CAGTCAAAGG	GAGAAAGAAG	ATGATAAAGT
451 GGAAGCCATA GTCCAATGGC	CATATGTCTG	GCAGGTCCTG	AAAGAGAATG
501 CTCTGACCCA GTGGACCTGG	CTGTGCCTTA	CCTACTCATA	TCTTTCTCAT
551 TAAAAGACTT ATGTAGAGAA	GAATTCAGGC	CTCATTGGAG	CCCTACTAGT
601 GGGAGTCTGG TTATACTACT	CCAAGGAAAA	GACACAGACC	TTGCACAAAT
651 TTTTGCTGTA ACAAAGAACT	TTTGATGAAG	GGAAAAGTTG	GCACTCAGAA
701 CCTTGATGCA GCCTAAAATG	GGATAGGGAT	GCTGCATCTG	CTCGGGCCTG
751 CACACAGTCA TGATTGGATG	ATGGTTATGT	AAACAGGTCT	CTGCCAGGTC
801 CCACAGGAAA ACCACTCCTG	TCAGTCTATT	GGCATGTGAT	TGGAATGGGC
851 AAGTGCACTC GAGGAACCAT	AATATTCCTC	GAAGGTCACA	CATTTCTTGT
901 CGCCAGGCGT CTGCTCAAAC	CCTTGGAAAT	CTCGCCAATA	ACTTTCCTTA
951 ACTCTTGATG ATCTCTTCCC	GACCTTGGAC	AGTTTCTACT	GTTTTGTCAT
1001 ACCAACATGA CTGTCCAGAG	TGGCATGGAA	GCTTATGTCA	AAGTAGACAG
1051 GAACCCCAAC ACTATGATGA	TACGAATGAA	AAATAATGAA	GAAGCGGAAG
1101 TGATCTTACT GATGACAACT	GATTCTGAAA	TGGATGTGGT	CAGGTTTGAT

- 193

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1151 CTCCTTCCTT TCCTAAAACT	TATCCAAATT	CGCTCAGTTG	CCAAGAAGCA
1201 TGGGTACATT ATGCTCCCTT	ACATTGCTGC	TGAAGAGGAG	GACTGGGACT
1251 AGTCCTCGCC TTGAACAATG	CCCGATGACA	GAAGTTATAA	AAGTCAATAT
1301 GCCCTCAGCG TATGGCATAC	GATTGGTAGG	AAGTACAAAA	AAGTCCGATT
1351 ACAGATGAAA AATCAGGAAT	CCTTTAAGAC	TCGTGAAGCT	ATTCAGCATG
1401 CTTGGGACCT TTGATTATAT	TTACTTTATG	GGGAAGTTGG	AGACACACTG
1451 TTAAGAATCA CGGAATCACT	AGCAAGCAGA	CCATATAACA	TCTACCCTCA
1501 GATGTCCGTC TAAAACATTT	CTTTGTATTC	AAGGAGATTA	CCAAAAGGTG
1551 GAAGGATTTT AAATGGACAG	CCAATTCTGC	CAGGAGAAAT	ATTCAAATAT
1601 TGACTGTAGA CCTGACCCGC	AGATGGGCCA	ACTAAATCAG	ATCCTCGGTG
1651 TATTACTCTA CAGGACTCAT	GTTTCGTTAA	TATGGAGAGA	GATCTAGCTT
1701 TGGCCCTCTC AGAGGAAACC	CTCATCTGCT	ACAAAGAATC	TGTAGATCAA
1751 AGATAATGTC ATTTGATGAG	AGACAAGAGG	AATGTCATCC	TGTTTTCTGT
1801 AACCGAAGCT TCCCCAATCC	GGTACCTCAC	AGAGAATATA	CAACGCTTTC
1851 AGCTGGAGTG AACATCATGC	CAGCTTGAGG	ATCCAGAGTT	CCAAGCCTCC
1901 ACAGCATCAA AGTTTGTTTG	TGGCTATGTT	TTTGATAGTT	TGCAGTTGTC
1951 CATGAGGTGG AGACTGACTT	CATACTGGTA	CATTCTAAGC	ATTGGAGCAC
2001 CCTTTCTGTC ATGGTCTATG	TTCTTCTCTG	GATATACCTT	CAAACACAAA
2051 AAGACACACT CTTCATGTCG	CACCCTATTC	CCATTCTCAG	GAGAAACTGT
2101 ATGGAAAACC CAGACTTTCG	CAGGTCTATG	GATTCTGGGG	TGCCACAACT
2151 GAACAGAGGC GACAAGAACA	ATGACCGCCT	TACTGAAGGT	TTCTAGTTGT
2201 CTGGTGATTA ATACTTGCTG	TTACGAGGAC	AGTTATGAAG	ATATTTCAGC
2251 AGTAAAAACA ACCCACCAGT	ATGCCATTGA	ACCAAGAAGC	TTCTCTCAAA
2301 CTTGAAACGC CAGTCAGATC	CATCAACGGG	AAATAACTCG	TACTACTCTT
2351 AAGAGGAAAT GAAGAAGGAA	TGACTATGAT	GATACCATAT	CAGTTGAAAT

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2401 GATTTTGACA GCAGCTTTCA	TTTATGATGA	GGATGAAAAT	CAGAGCCCCC
2451 AAAGAAAACA CTCTGGGATT	CGACACTATT	TTATTGCTGC	AGTGGAGAGG
2501 ATGGGATGAG TCAGAGTGGC	TAGCTCCCCA	CATGTTCTAA	GAAACAGGGC
2551 AGTGTCCCTC CTGATGGCTC	AGTTCAAGAA	AGTTGTTTTC	CAGGAATTTA
2601 CTTTACTCAG TTGGGACTCC	CCCTTATACC	GTGGAGAACT	AAATGAACAT
2651 TGGGGCCATA GGTAACTTTC	TATAAGAGCA	GAAGTTGAAG	ATAATATCAT
2701 AGAAATCAGG TTATTTCTTA	CCTCTCGTCC	CTATTCCTTC	TATTCTAGCC
2751 TGAGGAAGAT TTTGTCAAGC	CAGAGGCAAG	GAGCAGAACC	TAGAAAAAAC
2801 CTAATGAAAC TATGGCACCC	CAAAACTTAC	TTTTGGAAAG	TGCAACATCA
2851 ACTAAAGATG CTGATGTTGA	AGTTTGACTG	CAAAGCCTGG	GCTTATTTCT
2901 CCTGGAAAAA CTGGTCTGCC	GATGTGCACT	CAGGCCTGAT	TGGACCCCTT
2951 ACACTAACAC AGTACAGGAA	ACTGAACCCT	GCTCATGGGA	GACAAGTGAC
3001 TTTGCTCTGT GGTACTTCAC	TTTTCACCAT	CTTTGATGAG	ACCAAAAGCT
3051 TGAAAATATG CAGATGGAAG	GAAAGAAACT	GCAGGGCTCC	CTGCAATATC
3101 ATCCCACTTT TGGCTACATA	TAAAGAGAAT	TATCGCTTCC	ATGCAATCAA
3151 ATGGATACAC GGATTCGATG	TACCTGGCTT	AGTAATGGCT	CAGGATCAAA
3201 GTATCTGCTC ATTCATTTCA	AGCATGGGCA	GCAATGAAAA	CATCCATTCT
3251 GTGGACATGT AATGGCACTG	GTTCACTGTA	CGAAAAAAAG	AGGAGTATAA
3301 TACAATCTCT TACCATCCAA	ATCCAGGTGT	TTTTGAGACA	GTGGAAATGT
3351 AGCTGGAATT CTACATGCTG	TGGCGGGTGG	AATGCCTTAT	TGGCGAGCAT
3401 GGATGAGCAC GACTCCCCTG	ACTTTTTCTG	GTGTACAGCA	ATAAGTGTCA
3451 GGAATGGCTT CTTCAGGACA	CTGGACACAT	TAGAGATTTT	CAGATTACAG
3501 ATATGGACAG TCCGGATCAA	TGGGCCCCAA	AGCTGGCCAG	ACTTCATTAT
3551 TCAATGCCTG GGTGGATCTG	GAGCACCAAG	GAGCCCTTTT	CTTGGATCAA
3601 TTGGCACCAA CCCGTCAGAA	TGATTATTCA	CGGCATCAAG	ACCCAGGGTG

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3651 GTTCTCCAGC AGTCTTGATG	CTCTACATCT	CTCAGTTTAT	CATCATGTAT
3701 GGAAGAAGTG CTTAATGGTC	GCAGACTTAT	CGAGGAAATT	CCACTGGAAC
3751 TTCTTTGGCA TTTTTAACCC	ATGTGGATTC	ATCTGGGATA	AAACACAATA
3801 TCCAATTATT TATAGCATTC	GCTCGATACA	TCCGTTTGCA	CCCAACTCAT
3851 GCAGCACTCT TAGTTGCAGC	TCGCATGGAG	TTGATGGGCT	GTGATTTAAA
3901 ATGCCATTGG AGATTACTGC	GAATGGAGAG	TAAAGCAATA	TCAGATGCAC
3951 TTCATCCTAC TCAAAAGCTC	TTTACCAATA	TGTTTGCCAC	CTGGTCTCCT
4001 GACTTCACCT GGTGAATAAT	CCAAGGGAGG	AGTAATGCCT	GGAGACCTCA
4051 CCAAAAGAGT AAGTCACAGG	GGCTGCAAGT	GGACTTCCAG	AAGACAATGA
4101 AGTAACTACT TATGTGAAGG	CAGGGAGTAA	AATCTCTGCT	TACCAGCATG
4151 AGTTCCTCAT TCTCTTTTTT	CTCCAGCAGT	CAAGATGGCC	ATCAGTGGAC
4201 CAGAATGGCA CCTTCACACC	AAGTAAAGGT	TTTTCAGGGA	AATCAAGACT
4251 TGTGGTGAAC CTTCGAATTC	TCTCTAGACC	CACCGTTACT	GACTCGCTAC
4301 ACCCCCAGAG GGTTCTGGGC	TTGGGTGCAC	CAGATTGCCC	TGAGGATGGA
4351 TGCGAGGCAC CACCGTGCCC	AGGACCTCTA	CGACAAAACT	CACACATGCC
4401 AGCTCCAGAA CCCCCAAAAC	CTCCTGGGCG	GACCGTCAGT	CTTCCTCTTC
4451 CCAAGGACAC ATGCGTGGTG	CCTCATGATC	TCCCGGACCC	CTGAGGTCAC
4501 GTGGACGTGA GGTACGTGGA	GCCACGAAGA	CCCTGAGGTC	AAGTTCAACT
4551 CGGCGTGGAG GAGCAGTACA	GTGCATAATG	CCAAGACAAA	GCCGCGGGAG
4601 ACAGCACGTA CCAGGACTGG	CCGTGTGGTC	AGCGTCCTCA	CCGTCCTGCA
4651 CTGAATGGCA CCCTCCCAGC	AGGAGTACAA	GTGCAAGGTC	TCCAACAAAG
4701 CCCCATCGAG CGAGAACCAC	AAAACCATCT	CCAAAGCCAA	AGGGCAGCCC
4751 AGGTGTACAC GAACCAGGTC	CCTGCCCCCA	TCCCGGGATG	AGCTGACCAA
4801 AGCCTGACCT TCGCCGTGGA	GCCTGGTCAA	AGGCTTCTAT	CCCAGCGACA
4851 GTGGGAGAGC ACGCCTCCCG	AATGGGCAGC	CGGAGAACAA	CTACAAGACC

- 196

4901 TGTTGGACTC CGACGGCTCC TTCTTCCTCT ACAGCAAGCT

CACCGTGGAC

4951 AAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCG

TGATGCATGA

5001 GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA

5051 AATGA pSYNFVIII 010 protein sequence-(Dual chain FVIIIFc) (SEQ ID NO: 126)

2018203206 08 May 2018

1 MQIELSTCFF GELPVDARFP	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQSDL
51 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
101 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
151 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
201 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
251 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
301 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
351 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
401 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
451 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
501 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
551 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
601 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
651 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
701 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
751 SKNNAIEPRS DTISVEMKKE	FSQNPPVLKR	HQREITRTTL	QSDQEEIDYD
801 DFDIYDEDEN HVLRNRAQSG	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
851 SVPQFKKVVF EVEDNIMVTF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
901 RNQASRPYSF FWKVQHHMAP	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY
951 TKDEFDCKAW AHGRQVTVQE	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
1001 FALFFTIFDE YRFHAINGYI	TKSWYFTENM	ERNCRAPCNI	QMEDPTFKEN

- 197

2018203206 08 May 2018

1051 MDTLPGLVMA QDQRIRWYLL SMGSNENIHS IHFSGHVFTV

RKKEEYKMAL

1101 YNLYPGVFET VEMLPSKAGI WRVECLIGEH LHAGMSTLFL

VYSNKCQTPL

1151 GMASGHIRDF QITASGQYGQ WAPKLARLHY SGSINAWSTK EPFSWIKVDL

1201 LAPMIIHGIK TQGARQKFSS LYISQFIIMY SLDGKKWQTY RGNSTGTLMV

1251 FFGNVDSSGI KHNIFNPPII ARYIRLHPTH YSIRSTLRME LMGCDLNSCS

1301 MPLGMESKAI SDAQITASSY FTNMFATWSP SKARLHLQGR SNAWRPQVNN

1351 PKEWLQVDFQ KTMKVTGVTT QGVKSLLTSM YVKEFLISSS QDGHQWTLFF

1401 QNGKVKVFQG NQDSFTPVVN SLDPPLLTRY LRIHPQSWVH QIALRMEVLG

1451 CEAQDLYDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV

1501 VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW

1551 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV

1601 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

1651 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

Example 14: A new class of coagulation factor VIII molecules with greater than threefold half-life extension in hemophilia A mice [0380] The new class of FVIII molecules was designed to contain two polypeptides; one that consists of a single chain B-domain deleted (BDD) FVIII with XTEN inserted at one or more locations within the FVIII sequence, and one that is composed of the DD3 region of VWF. Each polypeptide was also recombinantly fused to the Fc region of IgGl to enable the DD3 region to be correctly aligned to bind the FVIII moiety. The resulting FVIII variants were expressed in HEK 293 cells by transient transfection, and purified from the conditioned media. FVIII activity was evaluated by FVIII chromogenic assay and the pharmacokinetic properties were assessed in both FVIII knockout (HemA) and FVIII/VWF double knock-out (DKO) mice.

[0381] Incorporating XTEN and D'D3 region of VWF into rFVIII led to the uncoupling of the clearance of the fusion proteins from endogenous VWF while extending their circulating half-life. FVIII in this fusion configuration is completely shielded from interacting with VWF, as measured by biolayer

- 198 2018203206 08 May 2018 [0382] interferometry (Octet) analysis. Consistent with this, their pharmacokinetic profiles in HemA and DKO mice were found to be identical, indicating that their clearance rate in mice was effectively disconnected from VWF. Optimization of XTEN length and the locations for inserting XTEN identified a subset of the proteins that have exceeded the VWF limitation (16 hours), reaching a circulating half-life of up to 30 hours in HemA mice representing a 4-fold improvement over BDD-FVIII. Importantly, these proteins maintained their functionality, as judged by FVIII chromogenic assay.

The VWF dependency has set a fundamental limitation for half-life of therapeutic FVIII. Uncoupling FVIII from VWF clearance pathways while extending half-life by the fusion of D'D3 region of VWF and XTEN has generated a novel FVIII molecule with a 4-fold half-life extension. This is the first report of an engineered FVIII that has exceeded the half-life limitation observed through industry-wide efforts in development of long-lasting FVIII, representing a potentially significant advancement in prophylactic treatment of hemophilia A.

Table 25: Protein sequences of FVIII-XTEN-Fc and VWF-Fc constructs FVIII 195 protein sequence (dual chain FVIIIFc with two 144 AE XTENs at amino acid 1656 and 1900) (SEQ ID NO: 105)

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVDARFP

51 PRVPKSFPFN	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
LGPTIQAEVY 101 DTVVITLKNM	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
EKEDDKVFPG 151 GSHTYVWQVL	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
LIGALLVCRE 201 GSLAKEKTQT	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
AASARAWPKM 251 HTVNGYVNRS	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
EGHTFLVRNH 301 RQASLEISPI	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
AYVKVDSCPE 351 EPQLRMKNNE	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
RSVAKKHPKT 401 WVHYIAAEEE	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
KYKKVRFMAY 451 TDETFKTREA	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
PYNIYPHGIT 501 DVRPLYSRRL	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
TKSDPRCLTR 551 YYSSFVNMER	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
NVILFSVFDE

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2018203206 08 May 2018

601 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
651 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
701 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
751 SKNNAIEPRS TASSSPGASP	FSQNPPVLKR	HQREITRTTL	QGAPGTPGSG
801 GTSSTGSPGA GTGPGTPGSG	SPGTSSTGSP	GASPGTSSTG	SPGSSPSAST
851 TASSSPGASP SPGSSTPSGA	GTSSTGSPGA	SPGTSSTGSP	GASPGTSSTG
901 TGSPGSSTPS DTISVEMKKE	GATGSPGASP	GTSSTGSPAS	SSDQEEIDYD
951 DFDIYDEDEN HVLRNRAQSG	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
1001 SVPQFKKVVF EVEDNIMVTF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
1051 RNQASRPYSF FWKVQHHMAP	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY
1101 TKDEFDCKAW AHGRQVTVQE	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
1151 FALFFTIFDE SEPATSGSET	TKSWYFTENM	ERNCRGAPTS	ESATPESGPG
1201 PGTSESATPE TEPSEGSAPG	SGPGSEPATS	GSETPGTSES	ATPESGPGTS
1251 TSESATPESG SPTSTEEGTS	PGSPAGSPTS	TEEGSPAGSP	TSTEEGSPAG
1301 ESATPESGPG YRFHAINGYI	TSTEPSEGSA	PGASSAPCNI	QMEDPTFKEN
1351 MDTLPGLVMA RKKEEYKMAL	QDQRIRWYLL	SMGSNENIHS	IHFSGHVFTV
1401 YNLYPGVFET VYSNKCQTPL	VEMLPSKAGI	WRVECLIGEH	LHAGMSTLFL
1451 GMASGHIRDF EPFSWIKVDL	QITASGQYGQ	WAPKLARLHY	SGSINAWSTK
1501 LAPMIIHGIK RGNSTGTLMV	TQGARQKFSS	LYISQFIIMY	SLDGKKWQTY
1551 FFGNVDSSGI LMGCDLNSCS	KHNIFNPPII	ARYIRLHPTH	YSIRSTLRME
1601 MPLGMESKAI SNAWRPQVNN	SDAQITASSY	FTNMFATWSP	SKARLHLQGR
1651 PKEWLQVDFQ QDGHQWTLFF	KTMKVTGVTT	QGVKSLLTSM	YVKEFLISSS
1701 QNGKVKVFQG QIALRMEVLG	NQDSFTPVVN	SLDPPLLTRY	LRIHPQSWVH
1751 CEAQDLYDKT SRTPEVTCVV	HTCPPCPAPE	LLGGPSVFLF	PPKPKDTLMI
1801 VDVSHEDPEV SVLTVLHQDW	KFNWYVDGVE	VHNAKTKPRE	EQYNSTYRVV

-200

1851 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV

1901 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS

FFLYSKLTVD

1951 KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK*

2018203206 08 May 2018

FVIII 196 protein sequence (dual chain FVIIIFc with three 144 AE XTENs at amino acid 26,1656 and 1900) (SEQ ID NO: 106)

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVGAPGS

51 SPSASTGTGP SSTGSPGSST	GSSPSASTGT	GPGASPGTSS	TGSPGASPGT
101 PSGATGSPGS GTGPGTPGSG	SPSASTGTGP	GASPGTSSTG	SPGSSPSAST
151 TASSSPGSST SPASSDARFP	PSGATGSPGS	STPSGATGSP	GASPGTSSTG
201 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
251 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
301 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
351 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
401 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
451 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
501 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
551 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
601 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
651 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
701 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
751 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
801 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
851 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
901 SKNNAIEPRS TASSSPGASP	FSQNPPVLKR	HQREITRTTL	QGAPGTPGSG
951 GTSSTGSPGA GTGPGTPGSG	SPGTSSTGSP	GASPGTSSTG	SPGSSPSAST
1001 TASSSPGASP SPGSSTPSGA	GTSSTGSPGA	SPGTSSTGSP	GASPGTSSTG

-201

2018203206 08 May 2018

1051 TGSPGSSTPS DTISVEMKKE	GATGSPGASP	GTSSTGSPAS	SSDQEEIDYD
1101 DFDIYDEDEN	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
HVLRNRAQSG 1151 SVPQFKKVVF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
EVEDNIMVTF 1201 RNQASRPYSF	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY
FWKVQHHMAP 1251 TKDEFDCKAW	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
AHGRQVTVQE 1301 FALFFTIFDE	TKSWYFTENM	ERNCRGAPTS	ESATPESGPG
SEPATSGSET 1351 PGTSESATPE	SGPGSEPATS	GSETPGTSES	ATPESGPGTS
TEPSEGSAPG 1401 TSESATPESG	PGSPAGSPTS	TEEGSPAGSP	TSTEEGSPAG
SPTSTEEGTS 1451 ESATPESGPG	TSTEPSEGSA	PGASSAPCNI	QMEDPTFKEN
YRFHAINGYI 1501 MDTLPGLVMA	QDQRIRWYLL	SMGSNENIHS	IHFSGHVFTV
RKKEEYKMAL 1551 YNLYPGVFET	VEMLPSKAGI	WRVECLIGEH	LHAGMSTLFL
VYSNKCQTPL 1601 GMASGHIRDF	QITASGQYGQ	WAPKLARLHY	SGSINAWSTK
EPFSWIKVDL 1651 LAPMIIHGIK	TQGARQKFSS	LYISQFIIMY	SLDGKKWQTY
RGNSTGTLMV 1701 FFGNVDSSGI	KHNIFNPPII	ARYIRLHPTH	YSIRSTLRME
LMGCDLNSCS 1751 MPLGMESKAI	SDAQITASSY	FTNMFATWSP	SKARLHLQGR
SNAWRPQVNN 1801 PKEWLQVDFQ	KTMKVTGVTT	QGVKSLLTSM	YVKEFLISSS
QDGHQWTLFF 1851 QNGKVKVFQG	NQDSFTPVVN	SLDPPLLTRY	LRIHPQSWVH
QIALRMEVLG 1901 CEAQDLYDKT	HTCPPCPAPE	LLGGPSVFLF	PPKPKDTLMI
SRTPEVTCVV 1951 VDVSHEDPEV	KFNWYVDGVE	VHNAKTKPRE	EQYNSTYRVV
SVLTVLHQDW 2001 LNGKEYKCKV	SNKALPAPIE	KTISKAKGQP	REPQVYTLPP
SRDELTKNQV 2051 SLTCLVKGFY	PSDIAVEWES	NGQPENNYKT	TPPVLDSDGS
FFLYSKLTVD 2101 KSRWQQGNVF	SCSVMHEALH	NHYTQKSLSL	SPGK*

FVIII 199 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 1656 and 1900) (SEQ ID NO: 107)

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL

GELPVDARFP

PRVPKSFPFN TSVVYKKTLF VEFTDHLFNI AKPRPPWMGL LGPTIQAEVY

-2022018203206 08 May 2018

101 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
151 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
201 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
251 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
301 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
351 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
401 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
451 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
501 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
551 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
601 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
651 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
701 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
751 SKNNAIEPRS TASSSPGASP	FSQNPPVLKR	HQAEITRTTL	QGAPGTPGSG
801 GTSSTGSPGA GTGPGTPGSG	SPGTSSTGSP	GASPGTSSTG	SPGSSPSAST
851 TASSSPGASP SPGSSTPSGA	GTSSTGSPGA	SPGTSSTGSP	GASPGTSSTG
901 TGSPGSSTPS DTISVEMKKE	GATGSPGASP	GTSSTGSPAS	SSDQEEIDYD
951 DFDIYDEDEN HVLRNRAQSG	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
1001 SVPQFKKVVF EVEDNIMVTF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
1051 RNQASRPYSF FWKVQHHMAP	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY
1101 TKDEFDCKAW AHGRQVTVQE	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
1151 FALFFTIFDE SEPATSGSET	TKSWYFTENM	ERNCRGAPTS	ESATPESGPG
1201 PGTSESATPE TEPSEGSAPG	SGPGSEPATS	GSETPGTSES	ATPESGPGTS
1251 TSESATPESG SPTSTEEGTS	PGSPAGSPTS	TEEGSPAGSP	TSTEEGSPAG
1301 ESATPESGPG YRFHAINGYI	TSTEPSEGSA	PGASSAPCNI	QMEDPTFKEN

-203

2018203206 08 May 2018

1351 MDTLPGLVMA RKKEEYKMAL	QDQRIRWYLL	SMGSNENIHS	IHFSGHVFTV
1401 YNLYPGVFET	VEMLPSKAGI	WRVECLIGEH	LHAGMSTLFL
VYSNKCQTPL 1451 GMASGHIRDF	QITASGQYGQ	WAPKLARLHY	SGSINAWSTK
EPFSWIKVDL 1501 LAPMIIHGIK	TQGARQKFSS	LYISQFIIMY	SLDGKKWQTY
RGNSTGTLMV 1551 FFGNVDSSGI	KHNIFNPPII	ARYIRLHPTH	YSIRSTLRME
LMGCDLNSCS 1601 MPLGMESKAI	SDAQITASSY	FTNMFATWSP	SKARLHLQGR
SNAWRPQVNN 1651 PKEWLQVDFQ	KTMKVTGVTT	QGVKSLLTSM	YVKEFLISSS
QDGHQWTLFF 1701 QNGKVKVFQG	NQDSFTPVVN	SLDPPLLTRY	LRIHPQSWVH
QIALRMEVLG 1751 CEAQDLYDKT	HTCPPCPAPE	LLGGPSVFLF	PPKPKDTLMI
SRTPEVTCVV 1801 VDVSHEDPEV	KFNWYVDGVE	VHNAKTKPRE	EQYNSTYRVV
SVLTVLHQDW 1851 LNGKEYKCKV	SNKALPAPIE	KTISKAKGQP	REPQVYTLPP
SRDELTKNQV 1901 SLTCLVKGFY	PSDIAVEWES	NGQPENNYKT	TPPVLDSDGS
FFLYSKLTVD 1951 KSRWQQGNVF	SCSVMHEALH	NHYTQKSLSL	SPGK*

FVIII 201 protein sequence (single chain FVIIIFc with three 144 AE XTENs at amino acid 26,1656 &1900) (SEQ ID NO: 108)

1 MQIELSTCFF GELPVGAPGS	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQSDL
51 SPSASTGTGP SSTGSPGSST	GSSPSASTGT	GPGASPGTSS	TGSPGASPGT
101 PSGATGSPGS GTGPGTPGSG	SPSASTGTGP	GASPGTSSTG	SPGSSPSAST
151 TASSSPGSST SPASSDARFP	PSGATGSPGS	STPSGATGSP	GASPGTSSTG
201 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
251 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
301 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
351 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
401 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
451 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
501 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI

-204

2018203206 08 May 2018

551 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
601 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
651 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
701 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
751 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
801 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
851 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
901 SKNNAIEPRS TASSSPGASP	FSQNPPVLKR	HQAEITRTTL	QGAPGTPGSG
951 GTSSTGSPGA GTGPGTPGSG	SPGTSSTGSP	GASPGTSSTG	SPGSSPSAST
1001 TASSSPGASP SPGSSTPSGA	GTSSTGSPGA	SPGTSSTGSP	GASPGTSSTG
1051 TGSPGSSTPS DTISVEMKKE	GATGSPGASP	GTSSTGSPAS	SSDQEEIDYD
1101 DFDIYDEDEN HVLRNRAQSG	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
1151 SVPQFKKVVF EVEDNIMVTF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
1201 RNQASRPYSF FWKVQHHMAP	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY
1251 TKDEFDCKAW AHGRQVTVQE	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
1301 FALFFTIFDE SEPATSGSET	TKSWYFTENM	ERNCRGAPTS	ESATPESGPG
1351 PGTSESATPE TEPSEGSAPG	SGPGSEPATS	GSETPGTSES	ATPESGPGTS
1401 TSESATPESG SPTSTEEGTS	PGSPAGSPTS	TEEGSPAGSP	TSTEEGSPAG
1451 ESATPESGPG YRFHAINGYI	TSTEPSEGSA	PGASSAPCNI	QMEDPTFKEN
1501 MDTLPGLVMA RKKEEYKMAL	QDQRIRWYLL	SMGSNENIHS	IHFSGHVFTV
1551 YNLYPGVFET VYSNKCQTPL	VEMLPSKAGI	WRVECLIGEH	LHAGMSTLFL
1601 GMASGHIRDF EPFSWIKVDL	QITASGQYGQ	WAPKLARLHY	SGSINAWSTK
1651 LAPMIIHGIK RGNSTGTLMV	TQGARQKFSS	LYISQFIIMY	SLDGKKWQTY
1701 FFGNVDSSGI LMGCDLNSCS	KHNIFNPPII	ARYIRLHPTH	YSIRSTLRME
1751 MPLGMESKAI SNAWRPQVNN	SDAQITASSY	FTNMFATWSP	SKARLHLQGR

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2018203206 08 May 2018

1801 PKEWLQVDFQ QDGHQWTLFF

1851 QNGKVKVFQG QIALRMEVLG

1901 CEAQDLYDKT SRTPEVTCVV 1951 VDVSHEDPEV SVLTVLHQDW 2001 LNGKEYKCKV SRDELTKNQV 2051 SLTCLVKGFY FFLYSKLTVD 2101 KSRWQQGNVF

KTMKVTGVTT

NQDSFTPVVN

HTCPPCPAPE

KFNWYVDGVE

SNKALPAPIE

PSDIAVEWES

SCSVMHEALH

QGVKSLLTSM

SLDPPLLTRY

LLGGPSVFLF

VHNAKTKPRE

KTISKAKGQP

NGQPENNYKT

NHYTQKSLSL

YVKEFLISSS

LRIHPQSWVH

PPKPKDTLMI

EQYNSTYRVV

REPQVYTLPP

TPPVLDSDGS

SPGK*

FVIII 203 protein sequence (single chain FVIIIFc with two AE XTENs; one

288AE XTEN in B-domain and one 144 AE XTEN at amino acid 1900) (SEQ ID NO: 109)

1 MQIELSTCFF GELPVDARFP	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQSDL
51 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
101 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
151 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
201 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
251 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
301 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
351 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
401 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
451 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
501 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
551 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
601 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
651 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
701 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
751 SKNNAIEPRS PGTSESATPE	FSQNGAPGTS	ESATPESGPG	SEPATSGSET
801 SGPGSEPATS SPAGSPTSTE	GSETPGTSES	ATPESGPGTS	TEPSEGSAPG

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2018203206 08 May 2018

851 EGTSESATPE AGSPTSTEEG	SGPGSEPATS	GSETPGTSES	ATPESGPGSP
901 SPAGSPTSTE	EGTSTEPSEG	SAPGTSESAT	PESGPGTSES
ATPESGPGTS 951 ESATPESGPG	SEPATSGSET	PGSEPATSGS	ETPGSPAGSP
TSTEEGTSTE 1001 PSEGSAPGTS	TEPSEGSAPG	SEPATSGSET	PGTSESATPE
SGPGTSTEPS 1051 EGSAPASSPP	VLKRHQAEIT	RTTLQSDQEE	IDYDDTISVE
MKKEDFDIYD 1101 EDENQSPRSF	QKKTRHYFIA	AVERLWDYGM	SSSPHVLRNR
AQSGSVPQFK 1151 KVVFQEFTDG	SFTQPLYRGE	LNEHLGLLGP	YIRAEVEDNI
MVTFRNQASR 1201 PYSFYSSLIS	YEEDQRQGAE	PRKNFVKPNE	TKTYFWKVQH
HMAPTKDEFD 1251 CKAWAYFSDV	DLEKDVHSGL	IGPLLVCHTN	TLNPAHGRQV
TVQEFALFFT 1301 IFDETKSWYF	TENMERNCRG	APTSESATPE	SGPGSEPATS
GSETPGTSES 1351 ATPESGPGSE	PATSGSETPG	TSESATPESG	PGTSTEPSEG
SAPGTSESAT 1401 PESGPGSPAG	SPTSTEEGSP	AGSPTSTEEG	SPAGSPTSTE
EGTSESATPE 1451 SGPGTSTEPS	EGSAPGASSA	PCNIQMEDPT	FKENYRFHAI
NGYIMDTLPG 1501 LVMAQDQRIR	WYLLSMGSNE	NIHSIHFSGH	VFTVRKKEEY
KMALYNLYPG 1551 VFETVEMLPS	KAGIWRVECL	IGEHLHAGMS	TLFLVYSNKC
QTPLGMASGH 1601 IRDFQITASG	QYGQWAPKLA	RLHYSGSINA	WSTKEPFSWI
KVDLLAPMII 1651 HGIKTQGARQ	KFSSLYISQF	IIMYSLDGKK	WQTYRGNSTG
TLMVFFGNVD 1701 SSGIKHNIFN	PPIIARYIRL	HPTHYSIRST	LRMELMGCDL
NSCSMPLGME 1751 SKAISDAQIT	ASSYFTNMFA	TWSPSKARLH	LQGRSNAWRP
QVNNPKEWLQ 1801 VDFQKTMKVT	GVTTQGVKSL	LTSMYVKEFL	ISSSQDGHQW
TLFFQNGKVK 1851 VFQGNQDSFT	PVVNSLDPPL	LTRYLRIHPQ	SWVHQIALRM
EVLGCEAQDL 1901 YDKTHTCPPC	PAPELLGGPS	VFLFPPKPKD	TLMISRTPEV
TCVVVDVSHE 1951 DPEVKFNWYV	DGVEVHNAKT	KPREEQYNST	YRVVSVLTVL
HQDWLNGKEY 2001 KCKVSNKALP	APIEKTISKA	KGQPREPQVY	TLPPSRDELT
KNQVSLTCLV 2051 KGFYPSDIAV	EWESNGQPEN	NYKTTPPVLD	SDGSFFLYSK
LTVDKSRWQQ 2101 GNVFSCSVMH	EALHNHYTQK	SLSLSPGK*

-207

2018203206 08 May 2018

FVIII 204 protein sequence (single chain FVIIIFc with two AE XTENs; one

288AE XTEN in B-domain and one 144 AE XTEN at amino acid 403) (SEQ ID NO: 110)

1 MQIELSTCFF GELPVDARFP	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQSDL
51 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
101 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
151 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
201 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
251 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
301 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
351 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
401 WVHYIAAEEE GSPTSTEEGT	DWDYAPLVLA	PDGAPTSTEP	SEGSAPGSPA
451 STEPSEGSAP SEGSAPGTSE	GTSTEPSEGS	APGTSESATP	ESGPGTSTEP
501 SATPESGPGS GSAPGTSESA	EPATSGSETP	GTSTEPSEGS	APGTSTEPSE
551 TPESGPGTSE KYKKVRFMAY	SATPESGPGA	SSDRSYKSQY	LNNGPQRIGR
601 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
651 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
701 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
751 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
801 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
851 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
901 SKNNAIEPRS PGTSESATPE	FSQNGAPGTS	ESATPESGPG	SEPATSGSET
951 SGPGSEPATS SPAGSPTSTE	GSETPGTSES	ATPESGPGTS	TEPSEGSAPG
1001 EGTSESATPE AGSPTSTEEG	SGPGSEPATS	GSETPGTSES	ATPESGPGSP
1051 SPAGSPTSTE ATPESGPGTS	EGTSTEPSEG	SAPGTSESAT	PESGPGTSES
1101 ESATPESGPG TSTEEGTSTE	SEPATSGSET	PGSEPATSGS	ETPGSPAGSP

-208

2018203206 08 May 2018

1151 PSEGSAPGTS SGPGTSTEPS	TEPSEGSAPG	SEPATSGSET	PGTSESATPE
1201 EGSAPASSPP	VLKRHQAEIT	RTTLQSDQEE	IDYDDTISVE
MKKEDFDIYD 1251 EDENQSPRSF	QKKTRHYFIA	AVERLWDYGM	SSSPHVLRNR
AQSGSVPQFK 1301 KVVFQEFTDG	SFTQPLYRGE	LNEHLGLLGP	YIRAEVEDNI
MVTFRNQASR 1351 PYSFYSSLIS	YEEDQRQGAE	PRKNFVKPNE	TKTYFWKVQH
HMAPTKDEFD 1401 CKAWAYFSDV	DLEKDVHSGL	IGPLLVCHTN	TLNPAHGRQV
TVQEFALFFT 1451 IFDETKSWYF	TENMERNCRA	PCNIQMEDPT	FKENYRFHAI
NGYIMDTLPG 1501 LVMAQDQRIR	WYLLSMGSNE	NIHSIHFSGH	VFTVRKKEEY
KMALYNLYPG 1551 VFETVEMLPS	KAGIWRVECL	IGEHLHAGMS	TLFLVYSNKC
QTPLGMASGH 1601 IRDFQITASG	QYGQWAPKLA	RLHYSGSINA	WSTKEPFSWI
KVDLLAPMII 1651 HGIKTQGARQ	KFSSLYISQF	IIMYSLDGKK	WQTYRGNSTG
TLMVFFGNVD 1701 SSGIKHNIFN	PPIIARYIRL	HPTHYSIRST	LRMELMGCDL
NSCSMPLGME 1751 SKAISDAQIT	ASSYFTNMFA	TWSPSKARLH	LQGRSNAWRP
QVNNPKEWLQ 1801 VDFQKTMKVT	GVTTQGVKSL	LTSMYVKEFL	ISSSQDGHQW
TLFFQNGKVK 1851 VFQGNQDSFT	PVVNSLDPPL	LTRYLRIHPQ	SWVHQIALRM
EVLGCEAQDL 1901 YDKTHTCPPC	PAPELLGGPS	VFLFPPKPKD	TLMISRTPEV
TCVVVDVSHE 1951 DPEVKFNWYV	DGVEVHNAKT	KPREEQYNST	YRVVSVLTVL
HQDWLNGKEY 2001 KCKVSNKALP	APIEKTISKA	KGQPREPQVY	TLPPSRDELT
KNQVSLTCLV 2051 KGFYPSDIAV	EWESNGQPEN	NYKTTPPVLD	SDGSFFLYSK
LTVDKSRWQQ 2101 GNVFSCSVMH	EALHNHYTQK	SLSLSPGK*

FVIII 205 protein sequence (single chain FVIIIFc with two AE XTENs; one

288AE XTEN in B-domain and one 144 AE XTEN at amino acid 18) (SEQ ID

NO: 111)

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP TSESATPESG

PGSEPATSGS ETPGTSESAT PESGPGSEPA TSGSETPGTS ESATPESGPG

101 TSTEPSEGSA PGSPAGSPTS TEEGTSESAT PESGPGSEPA TSGSETPGTS

-209 2018203206 08 May 2018

151 ESATPESGPG GELPVDARFP	SPAGSPTSTE	EGSPAGSPTS	TEEGASSSDL
201 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
251 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
301 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
351 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
401 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
451 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
501 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
551 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
601 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
651 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
701 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
751 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
801 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
851 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
901 SKNNAIEPRS PGTSESATPE	FSQNGAPGTS	ESATPESGPG	SEPATSGSET
951 SGPGSEPATS SPAGSPTSTE	GSETPGTSES	ATPESGPGTS	TEPSEGSAPG
1001 EGTSESATPE AGSPTSTEEG	SGPGSEPATS	GSETPGTSES	ATPESGPGSP
1051 SPAGSPTSTE ATPESGPGTS	EGTSTEPSEG	SAPGTSESAT	PESGPGTSES
1101 ESATPESGPG TSTEEGTSTE	SEPATSGSET	PGSEPATSGS	ETPGSPAGSP
1151 PSEGSAPGTS SGPGTSTEPS	TEPSEGSAPG	SEPATSGSET	PGTSESATPE
1201 EGSAPASSPP MKKEDFDIYD	VLKRHQAEIT	RTTLQSDQEE	IDYDDTISVE
1251 EDENQSPRSF AQSGSVPQFK	QKKTRHYFIA	AVERLWDYGM	SSSPHVLRNR
1301 KVVFQEFTDG MVTFRNQASR	SFTQPLYRGE	LNEHLGLLGP	YIRAEVEDNI
1351 PYSFYSSLIS HMAPTKDEFD	YEEDQRQGAE	PRKNFVKPNE	TKTYFWKVQH

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2018203206 08 May 2018

1401 CKAWAYFSDV TVQEFALFFT	DLEKDVHSGL	IGPLLVCHTN	TLNPAHGRQV
1451 IFDETKSWYF	TENMERNCRA	PCNIQMEDPT	FKENYRFHAI
NGYIMDTLPG 1501 LVMAQDQRIR	WYLLSMGSNE	NIHSIHFSGH	VFTVRKKEEY
KMALYNLYPG 1551 VFETVEMLPS	KAGIWRVECL	IGEHLHAGMS	TLFLVYSNKC
QTPLGMASGH 1601 IRDFQITASG	QYGQWAPKLA	RLHYSGSINA	WSTKEPFSWI
KVDLLAPMII 1651 HGIKTQGARQ	KFSSLYISQF	IIMYSLDGKK	WQTYRGNSTG
TLMVFFGNVD 1701 SSGIKHNIFN	PPIIARYIRL	HPTHYSIRST	LRMELMGCDL
NSCSMPLGME 1751 SKAISDAQIT	ASSYFTNMFA	TWSPSKARLH	LQGRSNAWRP
QVNNPKEWLQ 1801 VDFQKTMKVT	GVTTQGVKSL	LTSMYVKEFL	ISSSQDGHQW
TLFFQNGKVK 1851 VFQGNQDSFT	PVVNSLDPPL	LTRYLRIHPQ	SWVHQIALRM
EVLGCEAQDL 1901 YDKTHTCPPC	PAPELLGGPS	VFLFPPKPKD	TLMISRTPEV
TCVVVDVSHE 1951 DPEVKFNWYV	DGVEVHNAKT	KPREEQYNST	YRVVSVLTVL
HQDWLNGKEY 2001 KCKVSNKALP	APIEKTISKA	KGQPREPQVY	TLPPSRDELT
KNQVSLTCLV 2051 KGFYPSDIAV	EWESNGQPEN	NYKTTPPVLD	SDGSFFLYSK
LTVDKSRWQQ 2101 GNVFSCSVMH	EALHNHYTQK	SLSLSPGK*

pSYN FVIII 266 protein sequence (FVIII Fc with 42 AE-XTEN at amino acid 18 and 288 AE XTEN in B-domain) SEQ ID NO: 112

MQIELSTCFF GSPAGSPTST

EEGTSESATP FPPRVPKSFP

101 FNTSVVYKKT VYDTVVITLK

151 NMASHPVSLH PGGSHTYVWQ

201 VLKENGPMAS REGSLAKEKT

251 QTLHKFILLF KMHTVNGYVN

301 RSLPGLIGCH NHRQASLEIS

351 PITFLTAQTL PEEPQLRMKN

401 NEEAEDYDDD KTWVHYIAAE

LCLLRFCFSA

ESGPGSEPAT

LFVEFTDHLF

AVGVSYWKAS

DPLCLTYSYL

AVFDEGKSWH

RKSVYWHVIG

LMDLGQFLLF

LTDSEMDVVR

TRRYYLGAVE

SGSETPASSS

NIAKPRPPWM

EGAEYDDQTS

SHVDLVKDLN

SETKNSLMQD

MGTTPEVHSI

CHISSHQHDG

FDDDNSPSFI

LSWDYMQGAP

DLGELPVDAR

GLLGPTIQAE

QREKEDDKVF

SGLIGALLVC

RDAASARAWP

FLEGHTFLVR

MEAYVKVDSC

QIRSVAKKHP

-211

2018203206 08 May 2018

451 EEDWDYAPLV AYTDETFKTR	LAPDDRSYKS	QYLNNGPQRI	GRKYKKVRFM
501 EAIQHESGIL ITDVRPLYSR	GPLLYGEVGD	TLLIIFKNQA	SRPYNIYPHG
551 RLPKGVKHLK TRYYSSFVNM	DFPILPGEIF	KYKWTVTVED	GPTKSDPRCL
601 ERDLASGLIG DENRSWYLTE	PLLICYKESV	DQRGNQIMSD	KRNVILFSVF
651 NIQRFLPNPA CLHEVAYWYI	GVQLEDPEFQ	ASNIMHSING	YVFDSLQLSV
701 LSIGAQTDFL MSMENPGLWI	SVFFSGYTFK	HKMVYEDTLT	LFPFSGETVF
751 LGCHNSDFRN LLSKNNAIEP	RGMTALLKVS	SCDKNTGDYY	EDSYEDISAY
801 RSFSQNGAPG PESGPGSEPA	TSESATPESG	PGSEPATSGS	ETPGTSESAT
851 TSGSETPGTS TEEGTSESAT	ESATPESGPG	TSTEPSEGSA	PGSPAGSPTS
901 PESGPGSEPA EGSPAGSPTS	TSGSETPGTS	ESATPESGPG	SPAGSPTSTE
951 TEEGTSTEPS TSESATPESG	EGSAPGTSES	ATPESGPGTS	ESATPESGPG
1001 PGSEPATSGS TEPSEGSAPG	ETPGSEPATS	GSETPGSPAG	SPTSTEEGTS
1051 TSTEPSEGSA PSEGSAPASS	PGSEPATSGS	ETPGTSESAT	PESGPGTSTE
1101 PPVLKRHQAE YDEDENQSPR	ITRTTLQSDQ	EEIDYDDTIS	VEMKKEDFDI
1151 SFQKKTRHYF FKKVVFQEFT	IAAVERLWDY	GMSSSPHVLR	NRAQSGSVPQ
1201 DGSFTQPLYR SRPYSFYSSL	GELNEHLGLL	GPYIRAEVED	NIMVTFRNQA
1251 ISYEEDQRQG FDCKAWAYFS	AEPRKNFVKP	NETKTYFWKV	QHHMAPTKDE
1301 DVDLEKDVHS FTIFDETKSW	GLIGPLLVCH	TNTLNPAHGR	QVTVQEFALF
1351 YFTENMERNC PGLVMAQDQR	RAPCNIQMED	PTFKENYRFH	AINGYIMDTL
1401 IRWYLLSMGS PGVFETVEML	NENIHSIHFS	GHVFTVRKKE	EYKMALYNLY
1451 PSKAGIWRVE GHIRDFQITA	CLIGEHLHAG	MSTLFLVYSN	KCQTPLGMAS
1501 SGQYGQWAPK IIHGIKTQGA	LARLHYSGSI	NAWSTKEPFS	WIKVDLLAPM
1551 RQKFSSLYIS VDSSGIKHNI	QFIIMYSLDG	KKWQTYRGNS	TGTLMVFFGN
1601 FNPPIIARYI MESKAISDAQ	RLHPTHYSIR	STLRMELMGC	DLNSCSMPLG
1651 ITASSYFTNM LQVDFQKTMK	FATWSPSKAR	LHLQGRSNAW	RPQVNNPKEW

-212

2018203206 08 May 2018

1701 VTGVTTQGVK VKVFQGNQDS

1751 FTPVVNSLDP DLYDKTHTCP 1801 PCPAPELLGG

HEDPEVKFNW 1851 YVDGVEVHNA

EYKCKVSNKA 1901 LPAPIEKTIS

LVKGFYPSDI 1951 AVEWESNGQP

QQGNVFSCSV 2001 MHEALHNHYT

SLLTSMYVKE

PLLTRYLRIH

PSVFLFPPKP

KTKPREEQYN

KAKGQPREPQ

ENNYKTTPPV

QKSLSLSPGK

FLISSSQDGH

PQSWVHQIAL

KDTLMISRTP

STYRVVSVLT

VYTLPPSRDE

LDSDGSFFLY

QWTLFFQNGK

RMEVLGCEAQ

EVTCVVVDVS

VLHQDWLNGK

LTKNQVSLTC

SKLTVDKSRW pSYN FVIII 267 protein sequence (FVIII Fc with 72 AE-XTEN at amino acid 18 and 288

AE XTEN in B-domain) SEQ ID NO: 113

1 MQIELSTCFF TSESATPESG	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQGAP
51 PGSEPATSGS ESATPESGPG	ETPGTSESAT	PESGPGSEPA	TSGSETPGTS
101 TSTEPSEGSA VVYKKTLFVE	PGASSSDLGE	LPVDARFPPR	VPKSFPFNTS
151 FTDHLFNIAK HPVSLHAVGV	PRPPWMGLLG	PTIQAEVYDT	VVITLKNMAS
201 SYWKASEGAE NGPMASDPLC	YDDQTSQREK	EDDKVFPGGS	HTYVWQVLKE
251 LTYSYLSHVD KFILLFAVFD	LVKDLNSGLI	GALLVCREGS	LAKEKTQTLH
301 EGKSWHSETK GLIGCHRKSV	NSLMQDRDAA	SARAWPKMHT	VNGYVNRSLP
351 YWHVIGMGTT LTAQTLLMDL	PEVHSIFLEG	HTFLVRNHRQ	ASLEISPITF
401 GQFLLFCHIS EDYDDDLTDS	SHQHDGMEAY	VKVDSCPEEP	QLRMKNNEEA
451 EMDVVRFDDD DYAPLVLAPD	NSPSFIQIRS	VAKKHPKTWV	HYIAAEEEDW
501 DRSYKSQYLN HESGILGPLL	NGPQRIGRKY	KKVRFMAYTD	ETFKTREAIQ
551 YGEVGDTLLI GVKHLKDFPI	IFKNQASRPY	NIYPHGITDV	RPLYSRRLPK
601 LPGEIFKYKW ASGLIGPLLI	TVTVEDGPTK	SDPRCLTRYY	SSFVNMERDL
651 CYKESVDQRG FLPNPAGVQL	NQIMSDKRNV	ILFSVFDENR	SWYLTENIQR
701 EDPEFQASNI AQTDFLSVFF	MHSINGYVFD	SLQLSVCLHE	VAYWYILSIG
751 SGYTFKHKMV NSDFRNRGMT	YEDTLTLFPF	SGETVFMSME	NPGLWILGCH
801 ALLKVSSCDK QNGAPGTSES	NTGDYYEDSY	EDISAYLLSK	NNAIEPRSFS

-213

2018203206 08 May 2018

851 ATPESGPGSE ETPGTSESAT	PATSGSETPG	TSESATPESG	PGSEPATSGS
901 PESGPGTSTE PGSEPATSGS	PSEGSAPGSP	AGSPTSTEEG	TSESATPESG
951 ETPGTSESAT TSTEPSEGSA	PESGPGSPAG	SPTSTEEGSP	AGSPTSTEEG
1001 PGTSESATPE PATSGSETPG	SGPGTSESAT	PESGPGTSES	ATPESGPGSE
1051 SEPATSGSET PSEGSAPGSE	PGSPAGSPTS	TEEGTSTEPS	EGSAPGTSTE
1101 PATSGSETPG KRHQAEITRT	TSESATPESG	PGTSTEPSEG	SAPASSPPVL
1151 TLQSDQEEID KTRHYFIAAV	YDDTISVEMK	KEDFDIYDED	ENQSPRSFQK
1201 ERLWDYGMSS TQPLYRGELN	SPHVLRNRAQ	SGSVPQFKKV	VFQEFTDGSF
1251 EHLGLLGPYI EDQRQGAEPR	RAEVEDNIMV	TFRNQASRPY	SFYSSLISYE
1301 KNFVKPNETK EKDVHSGLIG	TYFWKVQHHM	APTKDEFDCK	AWAYFSDVDL
1351 PLLVCHTNTL NMERNCRAPC	NPAHGRQVTV	QEFALFFTIF	DETKSWYFTE
1401 NIQMEDPTFK LLSMGSNENI	ENYRFHAING	YIMDTLPGLV	MAQDQRIRWY
1451 HSIHFSGHVF GIWRVECLIG	TVRKKEEYKM	ALYNLYPGVF	ETVEMLPSKA
1501 EHLHAGMSTL GQWAPKLARL	FLVYSNKCQT	PLGMASGHIR	DFQITASGQY
1551 HYSGSINAWS SSLYISQFII	TKEPFSWIKV	DLLAPMIIHG	IKTQGARQKF
1601 MYSLDGKKWQ IIARYIRLHP	TYRGNSTGTL	MVFFGNVDSS	GIKHNIFNPP
1651 THYSIRSTLR SYFTNMFATW	MELMGCDLNS	CSMPLGMESK	AISDAQITAS
1701 SPSKARLHLQ TTQGVKSLLT	GRSNAWRPQV	NNPKEWLQVD	FQKTMKVTGV
1751 SMYVKEFLIS VNSLDPPLLT	SSQDGHQWTL	FFQNGKVKVF	QGNQDSFTPV
1801 RYLRIHPQSW PELLGGPSVF	VHQIALRMEV	LGCEAQDLYD	KTHTCPPCPA
1851 LFPPKPKDTL VEVHNAKTKP	MISRTPEVTC	VVVDVSHEDP	EVKFNWYVDG
1901 REEQYNSTYR IEKTISKAKG	VVSVLTVLHQ	DWLNGKEYKC	KVSNKALPAP
1951 QPREPQVYTL ESNGQPENNY	PPSRDELTKN	QVSLTCLVKG	FYPSDIAVEW
2001 KTTPPVLDSD LHNHYTQKSL 2051 SLSPGK*	GSFFLYSKLT	VDKSRWQQGN	VFSCSVMHEA

-214 pSYN FVIII 268 protein sequence (FVIII Fc with 144 AE-XTEN at amino

2018203206 08 May 2018

acid 18) SEO ID NO:	114
1 MQIELSTCFF TSESATPESG	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQGAP
51 PGSEPATSGS ESATPESGPG	ETPGTSESAT	PESGPGSEPA	TSGSETPGTS
101 TSTEPSEGSA TSGSETPGTS	PGSPAGSPTS	TEEGTSESAT	PESGPGSEPA
151 ESATPESGPG GELPVDARFP	SPAGSPTSTE	EGSPAGSPTS	TEEGASSSDL
201 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
251 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
301 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
351 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
401 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
451 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
501 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
551 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
601 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR
651 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
701 YYSSFVNMER NVILFSVFDE	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
751 NRSWYLTENI FDSLQLSVCL	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
801 HEVAYWYILS PFSGETVFMS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
851 MENPGLWILG SYEDISAYLL	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
901 SKNNAIEPRS DTISVEMKKE	FSQNPPVLKR	HQAEITRTTL	QSDQEEIDYD
951 DFDIYDEDEN HVLRNRAQSG	QSPRSFQKKT	RHYFIAAVER	LWDYGMSSSP
1001 SVPQFKKVVF EVEDNIMVTF	QEFTDGSFTQ	PLYRGELNEH	LGLLGPYIRA
1051 RNQASRPYSF FWKVQHHMAP	YSSLISYEED	QRQGAEPRKN	FVKPNETKTY
1101 TKDEFDCKAW AHGRQVTVQE	AYFSDVDLEK	DVHSGLIGPL	LVCHTNTLNP
1151 FALFFTIFDE YRFHAINGYI	TKSWYFTENM	ERNCRAPCNI	QMEDPTFKEN

-215

2018203206 08 May 2018

1201 MDTLPGLVMA RKKEEYKMAL	QDQRIRWYLL	SMGSNENIHS	IHFSGHVFTV
1251 YNLYPGVFET	VEMLPSKAGI	WRVECLIGEH	LHAGMSTLFL
VYSNKCQTPL 1301 GMASGHIRDF	QITASGQYGQ	WAPKLARLHY	SGSINAWSTK
EPFSWIKVDL 1351 LAPMIIHGIK	TQGARQKFSS	LYISQFIIMY	SLDGKKWQTY
RGNSTGTLMV 1401 FFGNVDSSGI	KHNIFNPPII	ARYIRLHPTH	YSIRSTLRME
LMGCDLNSCS 1451 MPLGMESKAI	SDAQITASSY	FTNMFATWSP	SKARLHLQGR
SNAWRPQVNN 1501 PKEWLQVDFQ	KTMKVTGVTT	QGVKSLLTSM	YVKEFLISSS
QDGHQWTLFF 1551 QNGKVKVFQG	NQDSFTPVVN	SLDPPLLTRY	LRIHPQSWVH
QIALRMEVLG 1601 CEAQDLYDKT	HTCPPCPAPE	LLGGPSVFLF	PPKPKDTLMI
SRTPEVTCVV 1651 VDVSHEDPEV	KFNWYVDGVE	VHNAKTKPRE	EQYNSTYRVV
SVLTVLHQDW 1701 LNGKEYKCKV	SNKALPAPIE	KTISKAKGQP	REPQVYTLPP
SRDELTKNQV 1751 SLTCLVKGFY	PSDIAVEWES	NGQPENNYKT	TPPVLDSDGS
FFLYSKLTVD 1801 KSRWQQGNVF	SCSVMHEALH	NHYTQKSLSL	SPGK*

pSYN FVIII 269 protein sequence (FVIII Fc with 72 AE-XTEN at amino acid

18) SEQ ID NO: 115

MQIELSTCFF TSESATPESG

PGSEPATSGS ESATPESGPG

101 TSTEPSEGSA VVYKKTLFVE

151 FTDHLFNIAK HPVSLHAVGV

201 SYWKASEGAE NGPMASDPLC

251 LTYSYLSHVD KFILLFAVFD

301 EGKSWHSETK GLIGCHRKSV

351 YWHVIGMGTT LTAQTLLMDL

401 GQFLLFCHIS EDYDDDLTDS

451 EMDVVRFDDD DYAPLVLAPD

501 DRSYKSQYLN HESGILGPLL

LCLLRFCFSA

ETPGTSESAT

PGASSSDLGE

PRPPWMGLLG

YDDQTSQREK

LVKDLNSGLI

NSLMQDRDAA

PEVHSIFLEG

SHQHDGMEAY

NSPSFIQIRS

NGPQRIGRKY

TRRYYLGAVE

PESGPGSEPA

LPVDARFPPR

PTIQAEVYDT

EDDKVFPGGS

GALLVCREGS

SARAWPKMHT

HTFLVRNHRQ

VKVDSCPEEP

VAKKHPKTWV

KKVRFMAYTD

LSWDYMQGAP

TSGSETPGTS

VPKSFPFNTS

VVITLKNMAS

HTYVWQVLKE

LAKEKTQTLH

VNGYVNRSLP

ASLEISPITF

QLRMKNNEEA

HYIAAEEEDW

ETFKTREAIQ

-216

2018203206 08 May 2018

551 YGEVGDTLLI GVKHLKDFPI	IFKNQASRPY	NIYPHGITDV	RPLYSRRLPK
601 LPGEIFKYKW ASGLIGPLLI	TVTVEDGPTK	SDPRCLTRYY	SSFVNMERDL
651 CYKESVDQRG FLPNPAGVQL	NQIMSDKRNV	ILFSVFDENR	SWYLTENIQR
701 EDPEFQASNI AQTDFLSVFF	MHSINGYVFD	SLQLSVCLHE	VAYWYILSIG
751 SGYTFKHKMV NSDFRNRGMT	YEDTLTLFPF	SGETVFMSME	NPGLWILGCH
801 ALLKVSSCDK QNPPVLKRHQ	NTGDYYEDSY	EDISAYLLSK	NNAIEPRSFS
851 AEITRTTLQS PRSFQKKTRH	DQEEIDYDDT	ISVEMKKEDF	DIYDEDENQS
901 YFIAAVERLW FTDGSFTQPL	DYGMSSSPHV	LRNRAQSGSV	PQFKKVVFQE
951 YRGELNEHLG SLISYEEDQR	LLGPYIRAEV	EDNIMVTFRN	QASRPYSFYS
1001 QGAEPRKNFV FSDVDLEKDV	KPNETKTYFW	KVQHHMAPTK	DEFDCKAWAY
1051 HSGLIGPLLV SWYFTENMER	CHTNTLNPAH	GRQVTVQEFA	LFFTIFDETK
1101 NCRAPCNIQM QRIRWYLLSM	EDPTFKENYR	FHAINGYIMD	TLPGLVMAQD
1151 GSNENIHSIH MLPSKAGIWR	FSGHVFTVRK	KEEYKMALYN	LYPGVFETVE
1201 VECLIGEHLH TASGQYGQWA	AGMSTLFLVY	SNKCQTPLGM	ASGHIRDFQI
1251 PKLARLHYSG GARQKFSSLY	SINAWSTKEP	FSWIKVDLLA	PMIIHGIKTQ
1301 ISQFIIMYSL NIFNPPIIAR	DGKKWQTYRG	NSTGTLMVFF	GNVDSSGIKH
1351 YIRLHPTHYS AQITASSYFT	IRSTLRMELM	GCDLNSCSMP	LGMESKAISD
1401 NMFATWSPSK MKVTGVTTQG	ARLHLQGRSN	AWRPQVNNPK	EWLQVDFQKT
1451 VKSLLTSMYV DSFTPVVNSL	KEFLISSSQD	GHQWTLFFQN	GKVKVFQGNQ
1501 DPPLLTRYLR CPPCPAPELL	IHPQSWVHQI	ALRMEVLGCE	AQDLYDKTHT
1551 GGPSVFLFPP NWYVDGVEVH	KPKDTLMISR	TPEVTCVVVD	VSHEDPEVKF
1601 NAKTKPREEQ KALPAPIEKT	YNSTYRVVSV	LTVLHQDWLN	GKEYKCKVSN
1651 ISKAKGQPRE DIAVEWESNG	PQVYTLPPSR	DELTKNQVSL	TCLVKGFYPS
1701 QPENNYKTTP SVMHEALHNH 1751 YTQKSLSLSP	PVLDSDGSFF GK*	LYSKLTVDKS	RWQQGNVFSC

-217

2018203206 08 May 2018 pSYNFVIII 271 protein sequence (FVIII Fc with 42 AE-XTEN at amino acid

18) SEQ ID NO: 116

MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQGAP GSPAGSPTST

EEGTSESATP ESGPGSEPAT SGSETPASSS DLGELPVDAR FPPRVPKSFP

101 FNTSVVYKKT LFVEFTDHLF NIAKPRPPWM GLLGPTIQAE VYDTVVITLK

151 NMASHPVSLH AVGVSYWKAS EGAEYDDQTS QREKEDDKVF PGGSHTYVWQ

201 VLKENGPMAS DPLCLTYSYL SHVDLVKDLN SGLIGALLVC REGSLAKEKT

251 QTLHKFILLF AVFDEGKSWH SETKNSLMQD RDAASARAWP KMHTVNGYVN

301 RSLPGLIGCH RKSVYWHVIG MGTTPEVHSI FLEGHTFLVR NHRQASLEIS

351 PITFLTAQTL LMDLGQFLLF CHISSHQHDG MEAYVKVDSC PEEPQLRMKN

401 NEEAEDYDDD LTDSEMDVVR FDDDNSPSFI QIRSVAKKHP KTWVHYIAAE

451 EEDWDYAPLV LAPDDRSYKS QYLNNGPQRI GRKYKKVRFM AYTDETFKTR

501 EAIQHESGIL GPLLYGEVGD TLLIIFKNQA SRPYNIYPHG ITDVRPLYSR

551 RLPKGVKHLK DFPILPGEIF KYKWTVTVED GPTKSDPRCL TRYYSSFVNM

601 ERDLASGLIG PLLICYKESV DQRGNQIMSD KRNVILFSVF DENRSWYLTE

651 NIQRFLPNPA GVQLEDPEFQ ASNIMHSING YVFDSLQLSV CLHEVAYWYI

701 LSIGAQTDFL SVFFSGYTFK HKMVYEDTLT LFPFSGETVF MSMENPGLWI

751 LGCHNSDFRN RGMTALLKVS SCDKNTGDYY EDSYEDISAY LLSKNNAIEP

801 RSFSQNPPVL KRHQAEITRT TLQSDQEEID YDDTISVEMK KEDFDIYDED

851 ENQSPRSFQK KTRHYFIAAV ERLWDYGMSS SPHVLRNRAQ SGSVPQFKKV

901 VFQEFTDGSF TQPLYRGELN EHLGLLGPYI RAEVEDNIMV TFRNQASRPY

951 SFYSSLISYE EDQRQGAEPR KNFVKPNETK TYFWKVQHHM APTKDEFDCK

1001 AWAYFSDVDL EKDVHSGLIG PLLVCHTNTL NPAHGRQVTV QEFALFFTIF

1051 DETKSWYFTE NMERNCRAPC NIQMEDPTFK ENYRFHAING YIMDTLPGLV

1101 MAQDQRIRWY LLSMGSNENI HSIHFSGHVF TVRKKEEYKM ALYNLYPGVF

1151 ETVEMLPSKA GIWRVECLIG EHLHAGMSTL FLVYSNKCQT PLGMASGHIR

-218

2018203206 08 May 2018

1201 DFQITASGQY DLLAPMIIHG

1251 IKTQGARQKF MVFFGNVDSS

1301 GIKHNIFNPP CSMPLGMESK

1351 AISDAQITAS NNPKEWLQVD

1401 FQKTMKVTGV FFQNGKVKVF

1451 QGNQDSFTPV LGCEAQDLYD

1501 KTHTCPPCPA VVVDVSHEDP

1551 EVKFNWYVDG DWLNGKEYKC

1601 KVSNKALPAP QVSLTCLVKG

1651 FYPSDIAVEW VDKSRWQQGN

1701 VFSCSVMHEA

GQWAPKLARL

SSLYISQFII

IIARYIRLHP

SYFTNMFATW

TTQGVKSLLT

VNSLDPPLLT

PELLGGPSVF

VEVHNAKTKP

IEKTISKAKG

ESNGQPENNY

LHNHYTQKSL

HYSGSINAWS

MYSLDGKKWQ

THYSIRSTLR

SPSKARLHLQ

SMYVKEFLIS

RYLRIHPQSW

LFPPKPKDTL

REEQYNSTYR

QPREPQVYTL

KTTPPVLDSD

SLSPGK*

TKEPFSWIKV

TYRGNSTGTL

MELMGCDLNS

GRSNAWRPQV

SSQDGHQWTL

VHQIALRMEV

MISRTPEVTC

VVSVLTVLHQ

PPSRDELTKN

GSFFLYSKLT pSYN FVIII protein sequence 272 (FVIII with 144 AE XTEN at amino acid and 244 AE XTEN in B-domain- no Fc) SEQ ID NO: 117

1 MQIELSTCFF TSESATPESG	LCLLRFCFSA	TRRYYLGAVE	LSWDYMQGAP
51 PGSEPATSGS ESATPESGPG	ETPGTSESAT	PESGPGSEPA	TSGSETPGTS
101 TSTEPSEGSA TSGSETPGTS	PGSPAGSPTS	TEEGTSESAT	PESGPGSEPA
151 ESATPESGPG GELPVDARFP	SPAGSPTSTE	EGSPAGSPTS	TEEGASSSDL
201 PRVPKSFPFN LGPTIQAEVY	TSVVYKKTLF	VEFTDHLFNI	AKPRPPWMGL
251 DTVVITLKNM EKEDDKVFPG	ASHPVSLHAV	GVSYWKASEG	AEYDDQTSQR
301 GSHTYVWQVL LIGALLVCRE	KENGPMASDP	LCLTYSYLSH	VDLVKDLNSG
351 GSLAKEKTQT AASARAWPKM	LHKFILLFAV	FDEGKSWHSE	TKNSLMQDRD
401 HTVNGYVNRS EGHTFLVRNH	LPGLIGCHRK	SVYWHVIGMG	TTPEVHSIFL
451 RQASLEISPI AYVKVDSCPE	TFLTAQTLLM	DLGQFLLFCH	ISSHQHDGME
501 EPQLRMKNNE RSVAKKHPKT	EAEDYDDDLT	DSEMDVVRFD	DDNSPSFIQI
551 WVHYIAAEEE KYKKVRFMAY	DWDYAPLVLA	PDDRSYKSQY	LNNGPQRIGR
601 TDETFKTREA PYNIYPHGIT	IQHESGILGP	LLYGEVGDTL	LIIFKNQASR

-219

2018203206 08 May 2018

651 DVRPLYSRRL TKSDPRCLTR	PKGVKHLKDF	PILPGEIFKY	KWTVTVEDGP
701 YYSSFVNMER	DLASGLIGPL	LICYKESVDQ	RGNQIMSDKR
NVILFSVFDE 751 NRSWYLTENI	QRFLPNPAGV	QLEDPEFQAS	NIMHSINGYV
FDSLQLSVCL 801 HEVAYWYILS	IGAQTDFLSV	FFSGYTFKHK	MVYEDTLTLF
PFSGETVFMS 851 MENPGLWILG	CHNSDFRNRG	MTALLKVSSC	DKNTGDYYED
SYEDISAYLL 901 SKNNAIEPRS	FSQNGAPGTS	ESATPESGPG	SEPATSGSET
PGTSESATPE 951 SGPGSEPATS	GSETPGTSES	ATPESGPGTS	TEPSEGSAPG
SPAGSPTSTE 1001 EGTSESATPE	SGPGSEPATS	GSETPGTSES	ATPESGPGSP
AGSPTSTEEG 1051 SPAGSPTSTE	EGTSTEPSEG	SAPGTSESAT	PESGPGTSES
ATPESGPGTS 1101 ESATPESGPG	SEPATSGSET	PGSEPATSGS	ETPGSPAGSP
TSTEEGTSTE 1151 PSEGSAPGTS	TEPSEGSAPG	SEPATSGSET	PGTSESATPE
SGPGTSTEPS 1201 EGSAPASSPP	VLKRHQAEIT	RTTLQSDQEE	IDYDDTISVE
MKKEDFDIYD 1251 EDENQSPRSF	QKKTRHYFIA	AVERLWDYGM	SSSPHVLRNR
AQSGSVPQFK 1301 KVVFQEFTDG	SFTQPLYRGE	LNEHLGLLGP	YIRAEVEDNI
MVTFRNQASR 1351 PYSFYSSLIS	YEEDQRQGAE	PRKNFVKPNE	TKTYFWKVQH
HMAPTKDEFD 1401 CKAWAYFSDV	DLEKDVHSGL	IGPLLVCHTN	TLNPAHGRQV
TVQEFALFFT 1451 IFDETKSWYF	TENMERNCRA	PCNIQMEDPT	FKENYRFHAI
NGYIMDTLPG 1501 LVMAQDQRIR	WYLLSMGSNE	NIHSIHFSGH	VFTVRKKEEY
KMALYNLYPG 1551 VFETVEMLPS	KAGIWRVECL	IGEHLHAGMS	TLFLVYSNKC
QTPLGMASGH 1601 IRDFQITASG	QYGQWAPKLA	RLHYSGSINA	WSTKEPFSWI
KVDLLAPMII 1651 HGIKTQGARQ	KFSSLYISQF	IIMYSLDGKK	WQTYRGNSTG
TLMVFFGNVD 1701 SSGIKHNIFN	PPIIARYIRL	HPTHYSIRST	LRMELMGCDL
NSCSMPLGME 1751 SKAISDAQIT	ASSYFTNMFA	TWSPSKARLH	LQGRSNAWRP
QVNNPKEWLQ 1801 VDFQKTMKVT	GVTTQGVKSL	LTSMYVKEFL	ISSSQDGHQW
TLFFQNGKVK 1851 VFQGNQDSFT	PVVNSLDPPL	LTRYLRIHPQ	SWVHQIALRM

EVLGCEAQDL 1901 Y*

-220 pSYN VWF 031 protein sequence (VWF Ρ1Ρ2Ρ'Ρ3- 48aa long thrombin cleavable GS

2018203206 08 May 2018 linker-Fc) SEQ ID NO: 118

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

201 ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL VDPEPFVALC

251 EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME

301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC

351 VHSGKRYPPG TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD

401 NRYFTFSGIC QYLLARDCQD HSFSIVIETV QCADDRDAVC TRSVTVRLPG

451 LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL RIQHTVTASV RLSYGEDLQM

501 DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG LAEPRVEDFG

551 NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS

601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL

651 NCPKGQVYLQ CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD

701 CVPKAQCPCY YDGEIFQPED IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD

751 AVLSSPLSHR SKRSLSCRPP MVKLVCPADN LRAEGLECTK TCQNYDLECM

801 SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE TVKIGCNTCV

851 CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS

901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE

951 THFEVVESGR YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD

1001 GIQNNDLTSS NLQVEEDPVD FGNSWKVSSQ CADTRKVPLD SSPATCHNNI

1051 MKQTMVDSSC RILTSDVFQD CNKLVDPEPY LDVCIYDTCS CESIGDCAAF

1101 CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY EAEWRYNSCA

-221

2018203206 08 May 2018

1151 PACQVTCQHP VDPEDCPVCE

1201 VAGRRFASGK ISGGGGSGGG 1251 GSGGGGSGGG

THTCPPCPAP 1301 ELLGGPSVFL

VKFNWYVDGV 1351 EVHNAKTKPR

VSNKALPAPI 1401 EKTISKAKGQ

YPSDIAVEWE 1451 SNGQPENNYK

FSCSVMHEAL 1501 HNHYTQKSLS

EPLACPVQCV

KVTLNPSDPE

GSGGGGSGGG

FPPKPKDTLM

EEQYNSTYRV

PREPQVYTLP

TTPPVLDSDG

LSPGK*

EGCHAHCPPG

HCQICHCDVV

GSLVPRGSGG

ISRTPEVTCV

VSVLTVLHQD

PSRDELTKNQ

SFFLYSKLTV

KILDELLQTC

NLTCEACQEP

GGSGGGGSDK

VVDVSHEDPE

WLNGKEYKCK

VSLTCLVKGF

DKSRWQQGNV pSYN VWF 034 protein sequence (VWF Ρ1Ρ2Ρ’Ρ3- 288AE XTEN- 35aa long thrombin cleavable GS linker-Fc) SEQ ID NO: 119

1 MIPARFAGVL	LALALILPGT	LCAEGTRGRS	STARCSLFGS
DFVNTFDGSM 51 YSFAGYCSYL	LAGGCQKRSF	SIIGDFQNGK	RVSLSVYLGE
FFDIHLFVNG 101 TVTQGDQRVS	MPYASKGLYL	ETEAGYYKLS	GEAYGFVARI
DGSGNFQVLL 151 SDRYFNKTCG	LCGNFNIFAE	DDFMTQEGTL	TSDPYDFANS
WALSSGEQWC 201 ERASPPSSSC	NISSGEMQKG	LWEQCQLLKS	TSVFARCHPL
VDPEPFVALC 251 EKTLCECAGG	LECACPALLE	YARTCAQEGM	VLYGWTDHSA
CSPVCPAGME 301 YRQCVSPCAR	TCQSLHINEM	CQERCVDGCS	CPEGQLLDEG
LCVESTECPC 351 VHSGKRYPPG	TSLSRDCNTC	ICRNSQWICS	NEECPGECLV
TGQSHFKSFD 401 NRYFTFSGIC	QYLLARDCQD	HSFSIVIETV	QCADDRDAVC
TRSVTVRLPG 451 LHNSLVKLKH	GAGVAMDGQD	IQLPLLKGDL	RIQHTVTASV
RLSYGEDLQM 501 DWDGRGRLLV	KLSPVYAGKT	CGLCGNYNGN	QGDDFLTPSG
LAEPRVEDFG 551 NAWKLHGDCQ	DLQKQHSDPC	ALNPRMTRFS	EEACAVLTSP
TFEACHRAVS 601 PLPYLRNCRY	DVCSCSDGRE	CLCGALASYA	AACAGRGVRV
AWREPGRCEL 651 NCPKGQVYLQ	CGTPCNLTCR	SLSYPDEECN	EACLEGCFCP
PGLYMDERGD 701 CVPKAQCPCY	YDGEIFQPED	IFSDHHTMCY	CEDGFMHCTM
SGVPGSLLPD 751 AVLSSPLSHR	SKRSLSCRPP	MVKLVCPADN	LRAEGLECTK
TCQNYDLECM

-222

2018203206 08 May 2018

801 SMGCVSGCLC TVKIGCNTCV	PPGMVRHENR	CVALERCPCF	HQGKEYAPGE
851 CRDRKWNCTD	HVCDATCSTI	GMAHYLTFDG	LKYLFPGECQ
YVLVQDYCGS 901 NPGTFRILVG	NKGCSHPSVK	CKKRVTILVE	GGEIELFDGE
VNVKRPMKDE 951 THFEVVESGR	YIILLLGKAL	SVVWDRHLSI	SVVLKQTYQE
KVCGLCGNFD 1001 GIQNNDLTSS	NLQVEEDPVD	FGNSWKVSSQ	CADTRKVPLD
SSPATCHNNI 1051 MKQTMVDSSC	RILTSDVFQD	CNKLVDPEPY	LDVCIYDTCS
CESIGDCAAF 1101 CDTIAAYAHV	CAQHGKVVTW	RTATLCPQSC	EERNLRENGY
EAEWRYNSCA 1151 PACQVTCQHP	EPLACPVQCV	EGCHAHCPPG	KILDELLQTC
VDPEDCPVCE 1201 VAGRRFASGK	KVTLNPSDPE	HCQICHCDVV	NLTCEACQEP
ISGTSESATP 1251 ESGPGSEPAT	SGSETPGTSE	SATPESGPGS	EPATSGSETP
GTSESATPES 1301 GPGTSTEPSE	GSAPGSPAGS	PTSTEEGTSE	SATPESGPGS
EPATSGSETP 1351 GTSESATPES	GPGSPAGSPT	STEEGSPAGS	PTSTEEGTST
EPSEGSAPGT 1401 SESATPESGP	GTSESATPES	GPGTSESATP	ESGPGSEPAT
SGSETPGSEP 1451 ATSGSETPGS	PAGSPTSTEE	GTSTEPSEGS	APGTSTEPSE
GSAPGSEPAT 1501 SGSETPGTSE	SATPESGPGT	STEPSEGSAP	DIGGGGGSGG
GGSLVPRGSG 1551 GDKTHTCPPC	PAPELLGGPS	VFLFPPKPKD	TLMISRTPEV
TCVVVDVSHE 1601 DPEVKFNWYV	DGVEVHNAKT	KPREEQYNST	YRVVSVLTVL
HQDWLNGKEY 1651 KCKVSNKALP	APIEKTISKA	KGQPREPQVY	TLPPSRDELT
KNQVSLTCLV 1701 KGFYPSDIAV	EWESNGQPEN	NYKTTPPVLD	SDGSFFLYSK
LTVDKSRWQQ 1751 GNVFSCSVMH	EALHNHYTQK	SLSLSPGK*

PSYNVWF036 protein sequence (VWF DlD2D^lD-98aa long thrombin cleavable GS linker-Fc) SEQ ID NO: 120

MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM

YSFAGYCSYL LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG

101 TVTQGDQRVS MPYASKGLYL ETEAGYYKLS GEAYGFVARI DGSGNFQVLL

151 SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL TSDPYDFANS WALSSGEQWC

-223

2018203206 08 May 2018

201 ERASPPSSSC VDPEPFVALC	NISSGEMQKG	LWEQCQLLKS	TSVFARCHPL
251 EKTLCECAGG CSPVCPAGME	LECACPALLE	YARTCAQEGM	VLYGWTDHSA
301 YRQCVSPCAR LCVESTECPC	TCQSLHINEM	CQERCVDGCS	CPEGQLLDEG
351 VHSGKRYPPG TGQSHFKSFD	TSLSRDCNTC	ICRNSQWICS	NEECPGECLV
401 NRYFTFSGIC TRSVTVRLPG	QYLLARDCQD	HSFSIVIETV	QCADDRDAVC
451 LHNSLVKLKH RLSYGEDLQM	GAGVAMDGQD	IQLPLLKGDL	RIQHTVTASV
501 DWDGRGRLLV LAEPRVEDFG	KLSPVYAGKT	CGLCGNYNGN	QGDDFLTPSG
551 NAWKLHGDCQ TFEACHRAVS	DLQKQHSDPC	ALNPRMTRFS	EEACAVLTSP
601 PLPYLRNCRY AWREPGRCEL	DVCSCSDGRE	CLCGALASYA	AACAGRGVRV
651 NCPKGQVYLQ PGLYMDERGD	CGTPCNLTCR	SLSYPDEECN	EACLEGCFCP
701 CVPKAQCPCY SGVPGSLLPD	YDGEIFQPED	IFSDHHTMCY	CEDGFMHCTM
751 AVLSSPLSHR TCQNYDLECM	SKRSLSCRPP	MVKLVCPADN	LRAEGLECTK
801 SMGCVSGCLC TVKIGCNTCV	PPGMVRHENR	CVALERCPCF	HQGKEYAPGE
851 CRDRKWNCTD YVLVQDYCGS	HVCDATCSTI	GMAHYLTFDG	LKYLFPGECQ
901 NPGTFRILVG VNVKRPMKDE	NKGCSHPSVK	CKKRVTILVE	GGEIELFDGE
951 THFEVVESGR KVCGLCGNFD	YIILLLGKAL	SVVWDRHLSI	SVVLKQTYQE
1001 GIQNNDLTSS SSPATCHNNI	NLQVEEDPVD	FGNSWKVSSQ	CADTRKVPLD
1051 MKQTMVDSSC CESIGDCAAF	RILTSDVFQD	CNKLVDPEPY	LDVCIYDTCS
1101 CDTIAAYAHV EAEWRYNSCA	CAQHGKVVTW	RTATLCPQSC	EERNLRENGY
1151 PACQVTCQHP VDPEDCPVCE	EPLACPVQCV	EGCHAHCPPG	KILDELLQTC
1201 VAGRRFASGK ISGGGGSGGG	KVTLNPSDPE	HCQICHCDVV	NLTCEACQEP
1251 GSGGGGSGGG GSGGGGSGGG	GSGGGGSGGG	GSGGGGSGGG	GSGGGGSGGG
1301 GSGGGGSGGG THTCPPCPAP	GSGGGGSGGG	GSLVPRGSGG	GGSGGGGSDK
1351 ELLGGPSVFL VKFNWYVDGV	FPPKPKDTLM	ISRTPEVTCV	VVDVSHEDPE
1401 EVHNAKTKPR VSNKALPAPI	EEQYNSTYRV	VSVLTVLHQD	WLNGKEYKCK

-224

1451 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF

YPSDIAVEWE

1501 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV

FSCSVMHEAL

1551 HNHYTQKSLS LSPGK*

2018203206 08 May 2018 pSYN Fc-015 protein sequence (IgG-Fc domain) SEQ ID NO: 121

METDTLLLWV FLFPPKPKDT

LMISRTPEVT PREEQYNSTY

101 RVVSVLTVLH GQPREPQVYT

151 LPPSRDELTK YKTTPPVLDS

201 DGSFFLYSKL LSLSPGK*

LLLWVPGSTG

CVVVDVSHED

QDWLNGKEYK

NQVSLTCLVK

TVDKSRWQQG

DKTHTCPPCP

PEVKFNWYVD

CKVSNKALPA

GFYPSDIAVE

NVFSCSVMHE

APELLGGPSV

GVEVHNAKTK

PIEKTISKAK

WESNGQPENN

ALHNHYTQKS

Example 15: FVIII-XTEN-Fc:VWF-Fc heterodimers have maintained normal FVIII specific activity as compared to wild type BDD-FVIII.

[0383] The FVIII specific activity of FVIII-XTEN-Fc:VWF-Fc heterodimers were determined. Heterodimers were purified using a two-step chromatography process. A weak anion exchange resin was used, followed by affinity chromatography. The final purified product had acceptable purity by SEC-HP LC. The specific activity was compared to B-domain deleted FVIII (BDD-FVIII), as measured by FVIII chromogenic assay and A280 concentration. The data are presented in Table 26. All tested molecules had demonstrated comparable FVIII specific activities to BDD-FVIII. Purity and the presence of each moiety of the molecules were confirmed by SDS-PAGE and western blotting.

Table 26: FVIII specific activity of FVIII-XTEN-Fc:VWF-Fc heterodimers

Construct	FVIII 207 scBDDFVIII	FVIII-66 dcBDD FVIII)	FVIII 155/ vWF31	FVIII 155/ vWF39	FVIII 169/ vWF31	FVIII 205/ vWF31	FVIII 169/ vWF34
Measured Specific Activity (IU/nmol)	1473	1592	1534	1796	1511	1345	1505

[0384] The half-lives of rFVIII-XTEN/D'D3 and BDD-FVIII were compared in

HemA Mice (Figure 15; Table 27). As Figure 15 shows, rFVIII-XTEN/D'D3

-225 achieved a half-life that was four fold longer than the half-life achieved by BDDFVIII.

2018203206 08 May 2018

Table 27: rFVIII-XTEN/D'D3 and BDD-FVIII in HemA mice

Treatment	5 minutes Recovery (%)	HL (hr)	MRT (hr)	Cl (mL/hr/kg)	Vss (mL/kg)	AUC D (hr*kg*mIU/mL/mIU)
BDD-FVIII	89	7.6	11	4.5	49.2	0.22
rFVIIIFc	78	16	20	2.9	57.8	0.35
rFVIII- XTEN/DO3	86	30	36	1.8	63.4	0.57

Example 16: FVIII-XTEN-Fc:VWF-Fc heterodimer's potency (FVIII activity) in hemostasis as measured by one stage aPTT assay [0385] The potency of FVIII-ΧΤΕΝ-Fc: VWF-Fc heterodimers in hemostasis was evaluated by their FVIII specific aPTT activity as summarized in Table 28. As demonstrated by Table 28, while the addition of the VWF D'D3 fragment and the insertion of XTEN into the intra-domains of FVIII reduce the FVIII specific aPTT activity of the heterodimers (as indicated by the FVIII155/VWF031 data and the FVIII205/VWF031 data), XTEN insertions in the FVIII B domain region or Cterminus of the VWF D'D3 fragment have no negative effect on the FVIII specific aPTT activity (as indicated by the FVIII 169/VWF031 data and the FVIII 169/VWF034 data). Compared to dual-chain BDD-FVIII (dcBDD-FVIII), FVIII155/VWF031, FVIII 169/VWF031, FVIII 169/VWF034 and VWF205/VWF031 showed reduction of specific aPTT activity by 2.5-fold, 2.8fold, 2.6-fold and 5.5-fold, respectively.

Table 28: FVIII specific aPTT activity of FVIII-XTEN-Fc:VWF-Fc heterodimers

Construct	FVIII 207 scBDDFVIII	FVIII-66 dcBDD- FVIII	FVIII 155/ VWF31	FVIII 169 /VWF31	FVIII 205 / VWF31	FVIII 169/ VWF34
Measured Specific aPTT Activity (IU/nmol)	818±153	1188 ± 213	448 ± 111	416 ±70	214 ±38	436±189

-2262018203206 08 May 2018 [0386] [0387]

FVIII specific aPTT assay

FVIII variants were diluted with aPTT buffer (0.15 M NaCl, 0.05 M TrisHC1, 1% BSA, pH 7.4) to the linear assay range (200- 1.6 mU/mL). 50 pL of diluted samples or standards were sequentially mixed with 50 pL of 37°C naive human HemA pooled plasma, 50 pL of 37°C aPTT reagent (ACTIN® FSL activated cephaloplastin reagent - Dade Behring, reference # B4219-2) and incubated at 37 °C for 4 minutes. 50 pi of 20 mM CaCh (Dade Behring [reference # ORFO37]) was then added to the reaction mixture to start the clotting reactions. Using the clotting time of each sample (the length of time from the addition of CaCh until the onset of clot formation), the aPTT activity was calculated against the standard that was generated with the 8^th international standard FVIII concentrate. Specific aPTT activity was calculated against the protein concentration of each molecule that measured by OD280.

Example 17: In vivo efficacy of FVIII-XTEN-Fc:VWF-Fc heterodimer in HemA mice Tail Clip bleeding model

To further access the hemostasis potency of the heterodimers, the acute efficacy of FVIII 169/VWF034 and FVIII205/VWF031 was evaluated in comparison with BDD-FVIII in the HemA mice Tail clip bleeding model. HemA mice were treated with a single IV injection of BDD-FVIII at 200, 65 and 20 IU/kg to generate the post tail clip injury blood loss control level. Blood loss from mice treated with 200 IU/kg of FVIII 169/VWF034 or FVIII205/VWF031 was compared to that of the BDD-FVIII treated control group mice to estimate their potency on hemostasis. Vehicle treated animals were used to generate blood loss baseline for the model. As shown in Figure 16, significant reduction in blood loss was observed from all FVIII treatment groups compared to that of the vehicle treated animals (p<0.05). Both FVIII 169/VWF034 and FVIII205/VWF031 are efficacious in the HemA mice Tail Clip model. Compared to BDD-FVIII, about 3 fold lower potency was observed for FVIII 169/VWF034, as demonstrated by the similar blood loss reduction achieved by 65 IU/kg BDD-FVIII and 200 IU/kg FVIII169/VWF034. As for FVIII205/VWF034, a 10 fold potency reduction has been observed, as demonstrated by the similar blood loss reduction achieved by 20 IU/kg BDD-FVIII and 200 IU/kg FVIII205/VWF031.

-2272018203206 08 May 2018 [0388] [0389] [0390] [0391]

Even though FVIII69/VWF034 and FVIII205/VWF031 had similar specific FVIII chromogenic activity compared to rBDD-FVIII, their FVIII aPTT activity and in vivo potency were both reduced due to the modifications of the molecules. Those data indicate that the aPTT activity of a FVIII molecule is a more accurate measurement on predicating its in vivo potency on hemostasis than the FVIII chromogenic activity.

HemA mice Tail clip bleeding model

8-10 weeks old male HemA mice were used for the study. Prior to tail clip injury, mice were anesthetized with a 50 mg/kg Ketamine/0.5 mg/kg Dexmedetomidine cocktail and placed on a 37°C heating pad to help maintain the body temperature. The tails of the mice were then be immersed in 37°C water for 10 minutes to dilate the lateral vein. After vein dilation, rFVIII or vehicle solution were injected via the tail vein and 5 min later, the distal 1 cm of the tail was cut off using a #11 scalpel with straight edge. The shed blood was collected into 13 ml of 37°C warm saline for 30 minutes and the mice were then euthanized while still under anesthesia by bilateral thoracotomy. Blood loss was quantified gravimetrically by weight change of the blood collection tubes before and after blood was collected in gram, which translated into milliliter (mL) of blood loss volume (lg weight change = 1 mL blood loss).

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as

-228 exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

All patents and publications cited herein are incorporated by reference herein in their entirety.

2018203206 08 May 2018 [0392]

-2292018203206 08 May 2018

Claims (7)

1. WHAT IS CLAIMED IS:

   1. A chimeric protein comprising (i) a von Willebrand Factor (VWF) protein comprising the D' domain and the D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein.

   2. A chimeric protein comprising a formula, which comprises:

   (a) V-X-FVIII, (b) FVIII-X-V, (c) V-X:FVIII, (d) X-V:FVIII, (e) FVIII:V-X, or (f) FVIII:X-V, wherein V comprises a VWF fragment,

   X comprises one or more XTEN sequences,

   FVIII comprises a FVIII protein;

   (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond or a non-covalent bond.

   3. The chimeric protein of claim 1 or 2, wherein the XTEN sequence is linked to the FVIII protein by a linker.

   4. The chimeric protein of claim 3, wherein the linker is a cleavable linker.

   5. The chimeric protein of any one of claims 1 to 4, which comprises a single polypeptide chain comprising the VWF fragment, the XTEN sequence and the FVIII protein.

   6. The chimeric protein of any one of claims 1 to 4, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises FVIII and the second polypeptide chain comprises the VWF fragment and the XTEN sequence.

   -230 2018203206 08 May 2018

   7. The chimeric protein of any one of claims 1 to 6, further comprising (iv) an Ig constant region or a portion thereof linked to either the VWF fragment, the XTEN sequence, or the FVIII protein, or any combination thereof.

   8. A chimeric protein comprising a formula, which comprises:

   (g) V-L2-X-L1-F1:FVIII-L3-F2;

   (h) V-L2-X-L1-F1:F2-L3-FVIII;

   (i) F1-L1-X-L2-V: FVIII-F3-F2;

   (j) F1-L1-X-L2-V:F2-L3-FVIII;

   (k) V-L2-X-L1-F1-L4-FVIII-L3-F2;

   (l) F2-L3-FVIII-L4-F1-L1-X-L2-V;

   (m) FVIII-L3-F2-L4-V-L2-X-L1-F1; and (n) F1-L1-X-L2-V-L4-F2-L3-FVIII, wherein V comprises a VWF fragment, each of Fl, F2, and F3 comprises an optional linker,

   F4 is an optional linker,

   FVIII comprises a FVIII protein,

   X comprises one or more XTEN sequences,

   Fl comprises an optional Ig constant region or a portion thereof,

   F2 comprises an optional additional Ig constant region or a portion thereof;

   (-) is a peptide bond or one or more amino acids; and (:) is a covalent bond or a non-covalent bond.

   9. The chimeric protein of any one of claims 7 or 8, wherein the Ig constant region or a portion thereof extends a half-life of the VWF fragment.

   10. The chimeric protein of any one of claims 7 to 9, wherein the Ig constant region or a portion thereof comprises a first Fc region, which is linked to the XTEN sequence or the VWF fragment.

   11. The chimeric protein of claim 10, wherein the Ig constant region or a portion thereof is linked to the XTEN sequence by a linker.

   12. The chimeric protein of claim 12, wherein the linker comprises a cleavable linker.

   -231 2018203206 08 May 2018

   13. The chimeric protein of any one of claims 7 to 12, further comprising an additional Ig constant region or a portion thereof.

   14. The chimeric protein of claim 13, wherein the additional Ig constant region or a portion thereof comprises an additional Fc region.

   15. The chimeric protein of claim 14, wherein the additional Ig constant region or a portion thereof extends the half-life of a FVIII protein.

   16. The chimeric protein of any one of claims 13 to 15, wherein the additional Ig constant region or a portion thereof is linked to the FVIII protein.

   17. The chimeric protein of claim 16, wherein the second Fc region is further linked to the VWF fragment by a linker.

   18. The chimeric protein of claim 17, wherein the linker is a processable linker.

   19. The chimeric protein of any one of claims 8 to 18, wherein L4 is a processable linker.

   20. The chimeric protein of any one of claims 7 to 19, wherein the additional Ig constant region or a portion thereof is associated with the Ig constant region or a portion thereof.

   21. The chimeric protein of claim 20, wherein the additional Ig constant region or a portion thereof is associated with the Ig constant region or a portion thereof by a covalent bond.

   22. The chimeric protein of claim 21, wherein the covalent bond is a disulfide bond.

   23. The chimeric protein of any one of claims 1 to 22, wherein the VWF fragment is associated with the FVIII protein by a non-covalent bond.

   24. The chimeric protein of any one of claims 1 to 23, wherein the half-life of the FVIII protein, is extended compared to a FVIII protein without the VWF fragment or compared to wild type FVIII.

   -232 2018203206 08 May 2018

   25. The chimeric protein of claim 24, wherein the half-life of the FVIII is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the VWF fragment or compared to wild type FVIII..

   26. The chimeric protein of claim 24, wherein the half-life of the Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours

   27. A chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, and (iii) an Ig constant region or a portion thereof, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or is inserted between two amino acids of at least one or more insertion sites in the FVIII protein and wherein the Ig constant region or a portion thereof is linked to or associated with the FVIII protein or the XTEN sequence.

   28. The chimeric protein of claim 27, wherein the XTEN sequence and the Ig constant region or a portion thereof extends the half-life of the FVIII protein.

   29. The chimeric protein of claim 27 or 28, wherein the VWF binding site is located in the A3 domain or the C2 domain of the FVIII protein or both the A3 domain and the C2 domain.

   30. The chimeric protein of claim 29, wherein the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO: 4.

   -233 2018203206 08 May 2018

   31. The chimeric protein of any one of claims 27 to 30, wherein the half-life of the FVIII protein is extended compared to a FVIII protein without the Ig constant region or a portion thereof or a FVIII protein without the XTEN sequence.

   32. The chimeric protein of claim 31, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than a FVIII protein without the Ig constant region or a portion thereof or a FVIII protein without the XTEN sequence or wild-type FVIII.

   33. The chimeric protein of claim 31, wherein the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

   34. The chimeric protein of any one of claims 27 to 33, wherein the Ig constant region or a portion thereof comprises a first Fc region.

   35. The chimeric protein of any one of claims 27 to 34, further comprising an additional Ig constant region or a portion thereof.

   36. The chimeric protein of claim 35, wherein the additional Ig constant region or a portion thereof comprises a second Fc region, which is linked to or associated with the first Fc region.

   37. The chimeric protein of claim 36, wherein the second Fc region is associated with the first Fc region by a covalent bond.

   38. The chimeric protein of claim 36, wherein the first Fc region is linked to the second Fc region by a linker.

   39. The chimeric protein of claim 38, wherein the linker is a processable linker.

   -234 2018203206 08 May 2018

   40. The chimeric protein of any one of claims 27 to 34, which comprises a single polypeptide chain comprising the FVIII protein, the XTEN sequence, and the lg constant region or a portion thereof.

   41. The chimeric protein of any one of claims 27 to 34, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein, the second polypeptide chain comprises a light chain of the FVIII protein and the XTEN sequence and the lg constant region or a portion thereof.

   42. The chimeric protein of any one of claims 35 to 39, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the FVIII protein, the XTEN sequence, and the lg constant region and the second polypeptide chain comprises the additional lg constant region or a portion thereof.

   43. The chimeric protein of any one of claims 35 to 39, which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein and the XTEN sequence, the second polypeptide chain comprises a light chain of the FVIII protein and the lg constant region or a portion thereof, and the third polypeptide chain comprises the additional lg constant region or a portion thereof.

   44. The chimeric protein of any one of claims 35 to 39, which comprises a single chain polypeptide comprising the FVIII protein, the XTEN sequence, the lg constant region or a portion thereof, and the additional lg constant region or a portion thereof.

   45. A chimeric protein comprising (i) a FVIII protein, (ii) an XTEN sequence, (iii) a VWF fragment, and (iv) an lg constant region or a portion thereof, which comprises the D' domain and the D3 domain of VWF, wherein the XTEN sequence is linked to the FVIII protein by an optional linker at the N-terminus or C terminus of the FVIII protein or inserted immediately downstream of one or more insertion sites in the FVIII protein, the VWF fragment is linked to or associated with the FVIII protein or the XTEN sequence, and the lg constant region or a portion thereof is linked to the FVIII protein, the XTEN sequence, the VWF fragment, or any combinations thereof.

   -235 2018203206 08 May 2018

   46. A chimeric protein comprising a formula, which comprises:

   (1) F VIII(X 1 )-L 1 -F1: V-L2-X2-L3 -F2;
2. (2) FVIII(X1)-L1-F1 :F2-L3-X2-L2-V;
3. (3) F1 -L1 -FVIII(X 1): V-L2-X2-L3 -F2;
4. (4) F1-L1-FVIII(X1);F2-L3-X2-L2-V;
5. (5) FVIII(X1)-L1 -F1-L4-V-L2-X2-L3-F2;
6. (6) FVIII(X1 )-L 1 -F1-L4-F2-L3-X2-L2-V;
7. (7) F1-L1-FVIII(X1)-L4-V-L2-X2-L3-F2, or (8) F1 -L1 -FVIII(X1 )-L4-F2-L3-X2-L2-V, wherein FVIII(Xl) comprises a FVIII protein and an XTEN sequence, wherein the XTEN sequence is linked to the N-terminus or C-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids (one or more insertion site) in the FVIII protein;

   each of LI, L2, or L3 comprises an optional linker;

   L4 is a linker;

   X2 comprises one or more XTEN sequences;

   FI comprises an Ig constant region or a portion thereof;

   F2 comprises an optional additional Ig constant region or a portion thereof, and V comprises a VWF fragment;

   (-) is a peptide bond or one or more amino acids; and (:) comprises a covalent bond or a non-covalent bond.

   47. The chimeric protein of claim 45 or 46, wherein the VWF fragment does not bind to a VWF clearance receptor.

   48. The chimeric protein of any one of claims 45 to 47, wherein the VWF fragment is capable of protecting the FVIII protein from cleavage by one or more protease, protecting the FVIII protein from activation, stabilizing the heavy chain and/or the light chain of the FVIII protein, or preventing clearance of the FVIII protein by one or more scavenger receptors.

   49. The chimeric protein of any one of claims 45 to 48, wherein the Ig constant region or a portion thereof inhibits or prevents endogenous VWF from binding to the FVIII protein by shielding or blocking a VWF binding site on the FVIII protein.

   -236 2018203206 08 May 2018

   50. The chimeric protein of claim 49, wherein the VWF binding site is located in the A3 domain or the C2 domain of the FVIII protein or both the A3 domain and the C2 domain.

   51. The chimeric protein of claim 49 or 50, wherein the VWF binding site comprises the amino acid sequence corresponding to amino acids 1669 to 1689 and 2303 to 2332 of SEQ ID NO: 4.

   52. The chimeric protein of any one of claims 45 to 51, wherein a half-life of the FVIII protein is extended compared to a FVIII protein without the VWF fragment.

   53. The chimeric protein of claim 52, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII.

   54. The chimeric protein of claim 52, wherein the half-life of the FVIII protein is at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 13 hours, at least about 14 hours, at least about 15 hours, at least about 16 hours, at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

   55. The chimeric protein of any one of claims 45 to 54, which comprises a single polypeptide chain comprising the FVIII protein, the XTEN sequence, the VWF fragment, and the Ig constant region or a portion thereof.

   56. The chimeric protein of any one of claims 45 to 54, wherein the Ig constant region or a portion thereof comprises a first Fc region.

   57. The chimeric protein of any one of claims 45 to 54, wherein the Ig constant region or a portion thereof is linked to the VWF fragment by an optional linker.

   58. The chimeric protein of claim 57, wherein the linker comprises a cleavable linker.

   -237 2018203206 08 May 2018

   59. The chimeric protein of claim 45 to 50, which further comprises an additional Ig constant region or a portion thereof, which is linked to the FVIII protein, the Ig constant region or a portion thereof, the VWF fragment, or any combinations thereof by an optional linker.

   60. The chimeric protein of claim 59, wherein the additional Ig constant region or a portion thereof is linked to the FVIII protein by an optional linker.

   61. The chimeric protein of claim 59 or 60, wherein the Ig constant region or a portion thereof is a second Fc region.

   62. The chimeric protein of any one of claims 8 to 26, 42 to 44, and 46 to 61, wherein the Ig constant region or a portion thereof and the additional Ig constant region or a portion thereof are identical or different.

   63. The chimeric protein of any one of claims 46 to 62, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof and the second polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof.

   64. The chimeric protein of any one of claims 46 to 62, which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein and the XTEN sequence, the second polypeptide chain comprises a light chain of the FVIII protein and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof.

   65. The chimeric protein of any one of claims 46 to 62, which comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein, the second polypeptide chain comprises a light chain of the FVIII protein, the XTEN sequence, and the Ig constant region or a portion thereof, and the third polypeptide chain comprises the VWF fragment and the additional Ig constant region or a portion thereof.

   -238 2018203206 08 May 2018

   66. The chimeric protein of any one of claims 46 to 62, wherein a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain of the FVIII protein and the XTEN sequence and the second polypeptide chain comprises a light chain of the FVIII protein, the Ig constant region or a portion thereof, the VWF fragment, and the additional Ig constant region or a portion thereof.

   67. The chimeric protein of any one of claims 63 to 66, which further comprises an additional XTEN sequence linked to the VWF fragment or to the additional Ig constant region or a portion thereof.

   68. The chimeric protein of any one of claims 1 to 26, wherein the FVIII protein is linked to an XTEN sequence at the C-terminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein or any combinations thereof.

   69. The chimeric protein of any one of claims 27 to 68, wherein the FVIII protein is linked to at least two XTEN sequences, at least three XTEN sequences, at least four XTEN sequences, at least five XTEN sequences, or at least six XTEN sequences.

   70. The chimeric protein of any one of claims 1 to 69, wherein the FVIII protein comprises one or more domains of FVIII selected from the group consisting of an A1 domain, al acidic region, an A2 domain, a2 acidic region, a B domain, an A3 domain, a3 acidic region, a Cl domain, a C2 domain, one or more fragments thereof, and any combinations thereof.

   71. The chimeric protein of any one of claims 27 to 70, wherein the one or more insertion sites in the FVIII protein is located within one or more domains of the FVIII protein selected from the group consisting of the Al domain, the al acidic region, the A2 domain, the a2 acidic region, the A3 domain, the B domain, the Cl domain, the C2 domain, and any combinations thereof or between one or more domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B domain and A3 domain, the A3 domain and Cl domain, the Cl domain and C2 domain, and any combinations thereof or between two domains of the FVIII protein selected from the group consisting of the Al domain and al acidic region, the al acidic region and A2 domain, the A2 domain and a2 acidic region, the a2 acidic region and B domain, the B

   -239 2018203206 08 May 2018 domain and A3 domain, the A3 domain and Cl domain, the Cl domain and C2 domain, and any combinations thereof.

   72. The chimeric protein of any one of claims 27 to 71, wherein the one or more insertion sites in the FVIII protein are one or more amino acids selected from the group consisting of the amino acid residues in Table 7, Table 8, Table 9 and Table 10.

   73. The chimeric protein of claim 72, wherein the XTEN sequence inserted at the insertion site corresponding to amino acids 3R of SEQ ID NO: 4 further comprises an amino acid sequence of ATR.

   74. The chimeric protein of any one of claims 27 to 71, wherein the one or more insertion sites in the FVIII protein are located immediately downstream of one or more amino acids selected from the group consisting of:

   (1) amino acid 3, (2) amino acid 18, (3) amino acid 22, (4) amino acid 26, (5) amino acid 32, (6) amino acid 40, (7) amino acid 60, (8) amino acid 65, (9) amino acid 81, (10) amino acid 116, (11) amino acid 119, (12) amino acid 130, (13) amino acid 188, (14) amino acid 211, (15) amino acid 216, (16) amino acid 220, (17) amino acid 224, (18) amino acid 230, (19) amino acid 333, (20) amino acid 336, (21) amino acid 339, (22) amino acid 375, (23) amino acid 399, (24) amino acid 403, (25) amino acid 409, (26) amino acid 416, (26) amino acid 442, (28) amino acid 487, (29) amino acid 490, (30) amino acid 494, (31) amino acid 500, (32) amino acid 518, (33) amino acid 599, (34) amino acid 603, (35) amino acid 713, (36) amino acid 745, (37) amino acid 1656, (38) amino acid 1711, (39) amino acid 1720, (40) amino acid 1725, (41) amino acid 1749, (42) amino acid 1796, (43) amino acid 1802, (44) amino acid 1827, (45) amino acid 1861, (46) amino acid 1896, (47) amino acid 1900, (48)

   amino acid 1904,

   -2402018203206 08 May 2018

   (49) amino acid 1905, amino acid 1937, (50) amino acid 1910, (51) (52) amino acid 2019, amino acid 2111, (53) amino acid 2068, (54) (55) amino acid 2120, amino acid 2188, (56) amino acid 2171, (57) (58) amino acid 2227, (59) amino acid 2277, and (60) two or more combinations thereof.

   75. The chimeric protein of any one of claims 1 to 74, wherein the FVIII protein comprises B domain or a portion thereof.

   76. The chimeric protein of claim 75, wherein the FVIII protein is SQ B domain deleted FVIII.

   77. The chimeric protein of any one of claims 1 to 76, wherein the FVIII protein comprises single chain FVIII.

   78. The chimeric protein of claim 77, wherein the single chain FVIII contains at least one amino acid substitution at a residue corresponding to residue 1648, residue 1645, or both of full-length mature Factor VIII polypeptide (SEQ ID NO: 4) or residue 754, residue 751, or both of SQ BDD Factor VIII (SEQ ID NO: 6).

   79. The chimeric protein of claim 78, wherein the amino acid substitution is an amino acid other than arginine.

   80. The chimeric protein of any one of claims 1 to 76, wherein the FVIII protein comprises a heavy chain of FVIII and a light chain of Factor VIII, wherein the heavy chain and the light chain are associated with each other by a metal bond.

   81. The chimeric protein of any one of claims 1 to 80, wherein the FVIII protein has a low affinity to or does not bind to a low-density lipoprotein receptor-related protein (LRP).

   82. The chimeric protein of claim 81, wherein the FVIII protein contains at least one amino acid substitution that lowers the affinity to or eliminates the binding to the LRP.

   83. The chimeric protein of claim 82, wherein the at least one amino acid substitution is at a residue corresponding to residue 471, residue 484, residue 487, residue 490, residue 497,

   -241 2018203206 08 May 2018 residue 2092, residue 2093 or two or more combinations thereof of full-length mature

   FVIII.

   84. The chimeric protein of claim 83, wherein the amino acid substitution at residue 471, 484, or 497 is an amino acid other than Arginine, the amino acid substitution at residue 487 is an amino acid other than Tyrosine, the amino acid substitution at residue 2092 is an amino acid other than Lysine, or the amino acid substitution at residue 2093 is an amino acid other than phenylalanine.

   85. The chimeric protein of any one of claims 1 to 84, wherein the FVIII protein contains at least one amino acid substitution, which induces the FVIII protein to be more stable than a FVIII protein without the substitution.

   86. The chimeric protein of claim 85, wherein the A2 domain and the A3 domain of the FVIII protein are associated to each other by a covalent bond.

   87. The chimeric protein of claim 85 or 86, wherein the at least one amino acid substitution is at a residue corresponding to residue 664, residue 1826, residue 662, or residue 1828 of full-length mature FVIII, or two or more combinations thereof.

   88. The chimeric protein of claim 87, wherein the FVIII protein contains (a) cysteine at the residue corresponding to residue 664 of full-length mature FVIII and cysteine at the residue corresponding to residue 1826 of full-length mature FVIII or (b) cysteine at the residue corresponding to residue 662 of full-length mature FVIII and cysteine at the residue corresponding to residue 1828 of full-length mature FVIII.

   89. The chimeric protein of any one of claims 1 to 26 and 45 to 88, wherein the VWF fragment is not amino acids 764 to 1274 of SEQ ID NO: 2.

   90. The chimeric protein of any one of claims 1 to 26 and 45 to 89, wherein the amino acid sequence of the D' domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2.

   91. The chimeric protein of any one of claims 1 to 26 and 45 to 90, wherein the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 867 to 1240 of SEQ ID NO: 2.

   -2422018203206 08 May 2018

   92. The chimeric protein of any one of claims 1 to 26 and 45 to 91, wherein the VWF fragment is a monomer.

   93. The chimeric protein of any one of claims 1 to 26 and 45 to 91, wherein the VWF fragment comprises at least two VWF fragments, at least three VWF fragments, at least four VWF fragments, at least five VWF fragments, or at least six VWF fragments.

   94. The chimeric protein of any one of claims 1 to 26 and 45 to 93, wherein the VWF fragment comprises an amino acid at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 1240 of SEQ ID NO: 2.

   95. The chimeric protein of claim 94, wherein the VWF fragment consists essentially of or consists of amino acids 764 to 1240 of SEQ ID NO: 2.

   96. The chimeric protein of any one of claims 1 to 26 and 45 to 95, wherein the VWF fragment contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2.

   97. The chimeric protein of claim 96, wherein the VWF fragment contains an amino acid other than cysteine substituted for a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2.

   98. The chimeric protein of any one of claims 1 to 26 and 45 to 97, wherein the VWF fragment further comprises the Dl domain, the D2 domain, or the Dl and D2 domains of VWF.

   99. The chimeric protein of any one of claims 1 to 27 and 45 to 98, wherein the VWF fragment further comprises a VWF domain selected from the group consisting of the Al domain, the A2 domain, the A3 domain, the D4 domain, the BI domain, the B2 domain, the B3 domain, the Cl domain, the C2 domain, the CK domain, one or more fragments thereof, and any combinations thereof

   100. The chimeric protein of any one of claims 1 to 27 and 45 to 99, wherein the VWF fragment consists essentially of or consists of: (1) the D' and D3 domains of VWF or fragments thereof; (2) the Dl, D', and D3 domains of VWF or fragments thereof; (3) the D2, D', and D3 domains of VWF or fragments thereof; (4) the Dl, D2, D', and D3

   -243 2018203206 08 May 2018 domains of VWF or fragments thereof; or (5) the Dl, D2, D', D3, and Al domains of

   VWF or fragments thereof

   101. The chimeric protein of any one of claims 1 to 27 and 45 to 100, wherein the VWF fragment further comprises a signal peptide of VWF or FVIII which is operably linked to the VWF fragment.

   102. The chimeric protein of any one of claims 1 to 101, wherein one or more of the linkers have a length of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acids.

   103. The chimeric protein of any one of claims 1 to 101, wherein one or more of the linkers have a length of about 1 to about 2000 amino acids.

   104. The chimeric protein of claim 103, wherein one or more of the linkers have a length of at least about 20, 35, 42, 48, 73, 75, 95, 98, 144, 288, 324, 333, 576, or 864 amino acids.

   105. The chimeric protein of any one of claims 1 to 104, wherein one or more of the linkers comprise a Gly/Ser peptide.

   106. The chimeric protein of claim 105, wherein the Gly/Ser peptide has a formula of (Gly4Ser)_n (SEQ ID NO: 139) or Ser(Gly4Ser)_n (SEQ ID NO: 140), wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

   107. The chimeric protein of claim 106, wherein the (Gly4Ser)_n linker is (Gly4Ser)3 (SEQ ID NO: 63) or (Gly₄Ser)₄ (SEQ ID NO: 138).

   108. The chimeric protein of any one of claims 1 to 107, wherein the XTEN sequence is selected from the group consisting of AE42, AE72, AE864, AE576, AE288, AE144, AG864, AG576, AG288, and AG144.

   109. The chimeric protein of claim 108, wherein the XTEN sequence is selected from the group consisting of SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO:

   -2442018203206 08 May 2018

   39; SEQ ID NO: 40; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 127.

   110. The chimeric protein of claim 109, wherein the XTEN sequence is AE288 or AG288.

   111. The chimeric protein of any one of claims 1 to 110, wherein the linker comprises at least one first cleavage site at the N-terminus of the linker, at least one second cleavage site at the C-terminus of the linker, or both.

   112. The chimeric protein of any one of claims 1 to 111, wherein the linker comprises 20 amino acids, 35 amino acids, 48 amino acids, 73 amino acids, or 95 amino acids.

   113. The chimeric protein of claim 112, wherein the linker is 48 amino acids thrombin cleavage linker.

   114. The chimeric protein of claim 112, wherein the linker is 35 amino acids thrombin cleavage linker.

   115. The chimeric protein of claim 111, wherein the one or more of the cleavage sites is TLDPRSFLLRNPNDKYEPFWEDEEK (SEQ ID NO: 8).

   116. The chimeric protein of claim 111, wherein one or more of the cleavage sites is cleaved by a protease selected from the group consisting of factor XIa, factor Xlla, kallikrein, factor Vila, factor IXa, factor Xa, factor Ila (thrombin), Elastase-2, GranzymeB, TEV, Enterokinase, Protease 3C, Sortase A, MMP-12, MMP-13, MMP-17, and MMP20.

   117. The chimeric protein of claim 111 or 116, wherein one or more of the cleavage sites comprise an amino acid sequence selected from the group consisting of RRRR (SEQ ID NO: 9), RKRRKR (SEQ ID NO: 10), RRRRS (SEQ ID NO: 11), TQSFNDFTR (SEQ ID NO: 12), SVSQTSKLTR (SEQ ID NO: 13), DFLAEGGGVR (SEQ ID NO: 14), TTKIKPR (SEQ ID NO: 15), LVPRG (SEQ ID NO: 16), ALRPR (SEQ ID NO: 17), KLTRAET (SEQ ID NO: 18), DFTRVVG (SEQ ID NO: 19), TMTRIVGG (SEQ ID NO: 20), SPFRSTGG (SEQ ID NO: 21), LQVRIVGG (SEQ ID NO: 22), PLGRIVGG (SEQ ID NO: 23), IEGRTVGG (SEQ ID NO: 24), LTPRSLLV (SEQ ID NO: 25), LGPVSGVP (SEQ ID NO: 26), VAGDSLEE (SEQ ID NO: 27), GPAGLGGA (SEQ ID NO: 28),

   -245 2018203206 08 May 2018

   GPAGLRGA (SEQ ID NO: 29), APLGLRLR (SEQ ID NO: 30), PALPLVAQ (SEQ ID

   NO: 31), ENLYFQG (SEQ ID NO: 32), DDDKIVGG (SEQ ID NO: 33), LEVLFQGP (SEQ ID NO: 34 ), and LPKTGSES (SEQ ID NO: 35).

   118. The chimeric protein of any one of claims 111 to 117, wherein the first cleavage site and the second cleavage site are identical or different.

   119. The chimeric protein of any one of claims 1 to 118, which is polysialylated, pegylated, or hesylated.

   120. A polynucleotide or a set of polynucleotides encoding the chimeric protein of any one of claims 1 to 120.

   121. The polynucleotide of claim 120, further comprising a polynucleotide chain, which encodes PC5 or PC7.

   122. A vector comprising the polynucleotide of claim 120 or 121 and one or more promoter operably linked to the polynucleotide or the set of polynucleotides.

   123. The vector of claim 122, further comprising an additional vector, which comprises a polynucleotide chain encoding PC5 or PC7.

   124. A host cell comprising the polynucleotide of any one of claims 120 or 121 or the vector of claim 122 or 123.

   125. The host cell of claim 124, which is a mammalian cell.

   126. The host cell of claim 125, wherein the mammalian cell is selected from the group consisting of HEK293 cell, CHO cell, and BHK cell.

   127. A pharmaceutical composition comprising the chimeric protein of any one of claims 1 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, or the host cell of any one of claims 124 to 125, and a pharmaceutically acceptable carrier.

   128. The composition of claim 127, wherein the FVIII protein has extended half-life compared to wild type FVIII protein.

   -2462018203206 08 May 2018

   129. The composition of claim 127 or 128, wherein the half-life of the FVIII protein is extended at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 11 times, or at least about 12 times longer than wild type FVIII.

   130. The composition of claim 128 or 129, wherein the half-life of Factor VIII is at least about 17 hours, at least about 18 hours, at least about 19 hours, at least about 20 hours, at least about 21 hours, at least about 22 hours, at least about 23 hours, at least about 24 hours, at least about 25 hours, at least about 26 hours, at least about 27 hours, at least about 28 hours, at least about 29 hours, at least about 30 hours, at least about 31 hours, at least about 32 hours, at least about 33 hours, at least about 34 hours, at least about 35 hours, at least about 36 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, or at least about 108 hours.

   131. The composition of any one of claims 127 to 130, which is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration.

   132. The composition of claim 131, wherein the parenteral administration is intravenous or subcutaneous administration.

   133. The composition of any one of claims 127 to 132, which is used to treat a bleeding disease or condition in a subject in need thereof.

   134. The composition of claim 133, wherein the bleeding disease or condition is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, bleeding in the illiopsoas sheath and any combinations thereof.

   -2472018203206 08 May 2018

   135. The composition of claim 133 or 134, wherein the subject is scheduled to undergo a surgery.

   136. The composition of any one of claims 133 to 135, wherein the treatment is prophylactic or on-demand.

   137. A method of preventing or inhibiting binding of a FVIII protein with endogenous VWF comprising adding an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus prevents or inhibits binding of endogenous VWF.

   138. A method of extending or increasing half-life of a FVIII protein, wherein the method comprises adding an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases half-life of the FVIII protein.

   139. A method of preventing or inhibiting clearance of a FVIII protein from a cell, wherein the method comprises adding an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 to a cell comprising a FVIII protein or a polynucleotide encoding the FVIII protein, wherein the protein having VWF activity binds to the FVIII protein.

   140. The method of any one of claims 137 to 139, wherein the subject is an animal.

   141. The method of claim 140, wherein the animal is a human.

   142. The method of claim 140, wherein the subject is suffering from hemophilia A.

   143. A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein of any one of claims 1 to 26 and 45 to 119, the polynucleotide of claim 120 or 121, the vector of claim

   -248 2018203206 08 May 2018

   122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136, wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the illiopsoas sheath.

   144. The method of claim 143, wherein the treatment is prophylactic or on-demand.

   145. The method of claim 143 or 144, wherein the effective amount is 0.1 pg/kg to 500 mg/kg.

   146. The method of any one of claims 143 to 145, wherein the chimeric protein of any one of claims 1 to 119, the polynucleotide of claim 120 or 121, the vector of claim 122 or 123, the host cell of any one of claims 124 to 126, or the composition of any one of claims 127 to 136 is administered by a route selected from the group consisting of topical administration, intraocular administration, parenteral administration, intrathecal administration, subdural administration and oral administration.

   147. The method of claim 146, wherein the parenteral administration is selected from the group consisting of intravenous administration, subcutaneous administration, intramuscular administration, and intradermal administration,

   148. A method of making a chimeric protein, comprising transfecting one or more host cell with the polynucleotide of claim 120 or 121 or the vector of claim 122 or 123 and expressing the chimeric protein in the host cell.

   149. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion site is immediately downstream of residue 745 corresponding to the mature FVIII protein (SEQ ID NO: 4).

   150. The chimeric protein of any one of claims 27-119, where the XTEN insertion sites are immediately downstream of residue 1656 and residue 1900 of the FVIII protein.

   -2492018203206 08 May 2018

   151. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 26, 1656, and 1900 of the FVIII protein.

   152. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 403 and 745 of the FVIII protein.

   153. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 745 and 1900 of the FVIII protein.

   154. The chimeric protein of any one of claims 27-119, wherein the XTEN insertion sites are immediately downstream of residues 18 and 745 of the FVIII protein.

   155. The chimeric protein of any one of claims 149-154, wherein the FVIII protein is a dual chain FVIII isoform.

   156. The chimeric protein of any one of claims 149-154, wherein the FVIII protein is a single chain FVIII isoform.

   157. The chimeric protein of any one of claims 149-156, comprising one XTEN.

   158. The chimeric protein of any one of claims 149-156, comprising two XTENs.

   159. The chimeric protein of any one of claims 149-156, comprising three XTENS are inserted.

   160. The chimeric protein of any one of claims 149-157, wherein the XTEN is SEQ ID NO: 39 (AE288).

   161. The chimeric protein of any one of claims 149-156 and 158, wherein the XTENs are SEQ ID NOs: 38 and 37 (AG144 and AE144).

   162. The chimeric protein of any one of claims 149-156, and 159 wherein the XTENs are SEQ ID NOs: 37, 38 and 37 (AE144, AG144, and AE144).

   163. The chimeric protein of any one of claims 149-156 and 157, wherein the XTENs are SEQ ID NOs: 38 and 39(AE144 and AE288).

   164. The host cell of claim 124, wherein the PC5 or PC7 cleaves the D1D2 domains of VWF.

   -250 165. The chimeric protein of any one of claims 1 to 27, which comprises at least two

   XTENs, at least three XTENs, at least four XTENs, at least five XTENs, or at least six

   XTENs.

   2018203206 08 May 2018

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   97047 1 SEQUENCE LI STI NG <110> Bi ogen I dec MA I nc. Chhabr a, Ekt a Set h Li u, Tongyao <120> FACTOR VI I I COMPLEX WITH XTEN AND VON WILLEBRAND FACTOR PROTEI N, AND USES THEREOF <130> 2159. 365PC06/ EKS/ C- K/ E- H <140> To be assi gned <141> Her ewi t h <150> US 61/840, 811 <151> 2013- 06- 28 <150> US 61/ 827, 158 <151> 2013- 05- 24 <150> US 61/ 801, 544 <151> 2013- 03- 15 <150> US 61/ 801, 504 <151> 2013- 03- 15 <150> US 61/ 759, 819 <151> 2013- 02- 01 <150> US 61/ 670, 401 <151> 2012- 07- 11 <160> 140

   Page 1

   2018203206 08 May 2018

   97047_1 <170> Pat ent I n version 3.5

   <210> 1 <211> 8442 <212> DNA <213> Homo sapi ens <400> 1

   at gat t cct g ct t t gt gcag gact t cgt ca ct ggcagggg agagt gagcc accgt gacac gaaact gagg gat ggcagcg ct gt gt ggca acct cggacc gaacgggcat ct gt gggagc gt ggaccccg ccagat t t gc aaggaact cg acacct t t ga gct gccagaa t ct ccgt gt a agggggacca ct gggt act a gcaact t t ca act t t aacat ct t at gact t ct cct cccag agt gccagct agcct t t t gt cggggt gct g cggcaggt ca t gggagcat g acgct cct t c tcttggggaa aagagt ct cc caagct gt cc agt cct gct g ct 11 gct gaa t gccaact ca cagct cat gc t ct gaagagc ggccct gt gt ct t gct ct gg t ccacggccc t acagct 11 g t cgat t at t g t t t t t t gaca at gccct at g ggt gaggcct t cagacagat gat gact t t a t gggct ct ga aacat ct cct acct cggt gt gagaagact t ccct cat t t t gat gcagcct cgggat act g gggact t cca t ccat 11 gt t cct ccaaagg at ggct t t gt act t caacaa t gacccaaga gcagt ggaga ct ggggaaat

   11 gcccgct g t gt gt gagt g gccagggacc t t t cggaagt cagt t acct c gaat ggcaag t gt caat ggt gct gt at ct a ggccaggat c gacct gcggg agggacct t g acagt ggt gt gcagaagggc ccaccct ct g tgctgggggg

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   2018203206 08 May 2018 ct ggagt gcg gt gct gt acg t at aggcagt t gt caggagc ct ct gcgt gg acct ccct ct aat gaagaat aacagat act cact cct t ct acccgct ccg ggggcaggag cgcat ccagc gact gggat g t gcggcct gt ct ggcrgagc gacct gcaga gaggaggcgt cct gccct gc gct ggaccga gt gt gt cccc gat gcgt gga agagcaccga ct cgagact g gt ccagggga t cacct t cag ccat t gt cat t caccgt ccg t t gccat gga at acagt gac gccgcgggag gt gggaat t a cccgggt gga agcagcacag gcgcggt cct cct cct ggag ccacagcgcg tt gcgccagg t ggct gcagc gt gt ccct gc caacacct gc gt gcct t gt c t gggat ct gc t gagact gt c gct gcct ggc t ggccaggac ggcct ccgt g gct gct ggt g caat ggcaac ggactt cggg cgat ccct gc gacgt ccccc

   97047_1 t acgcccgga t gcagcccag acct gccaga t gccct gagg gt gcat t ccg at t t gccgaa act ggt caat cagt acct gc cagt gt gct g ct gcacaaca at ccagct cc cgcct cagct aagct gt ccc cagggcgacg aacgcct gga gccct caacc acat t cgagg cct gt gccca t gt gccct gc gcct gcacat gacagct cct gaaagcgct a acagccagt g cccact t caa t ggcccggga at gaccgcga gcct t gt gaa ccct cct gaa acggggagga ccgt ct at gc act t cct t ac agct gcacgg cgcgcat gac cct gccat cg ggagggaat g t ggt at ggag caat gaaat g ggat gaaggc ccct cccggc gat ct gcagc gagct t t gac t t gccaggac cgct gt gt gc act gaagcat aggt gacct c cct gcagat g cgggaagacc cccct ct ggg ggact gccag caggt t ct cc t gccgt cagc

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   2018203206 08 May 2018 ccgct gccct t gcct gt gcg gcgt ggcgcg t gcgggaccc gaggcct gcc t gcgt gccca at ct t ct cag agt ggagt cc agcaaaagga ct gcgggct g agcat gggct t gt gt ggccc acagt gaaga cat gt gt gt g ct caaat acc aaccct ggga t gcaagaaac acct gcggaa gcgccct ggc agccaggccg cct gcaacct t ggagggct g aggcccagt g accat cacac ccggaagct t gcct at cct g aagggct cga gt gt ct ct gg t ggaaaggt g t t ggct gcaa at gccacgt g t gt t ccccgg cct t t cggat gggt caccat ct gccgct ac cagct at gcc ct gt gagct g gacct gccgc ct t ct gcccc cccct gt t ac cat gt gct ac gct gcct gac t cggcccccc gt gt accaaa ct gcct ct gc t ccct gct t c cact t gt gt c ct ccacgat c ggagt gccag cct agt gggg cct ggt ggag

   97047_1 gacgt gt gct gcggcct gcg aact gcccga t ct ct ct ct t ccagggct ct t at gacggt g t gt gaggat g gct gt cct ca at ggt caagc acgt gccaga cccccgggca cat cagggca t gt cgggacc ggcat ggccc t acgt t ct gg aat aagggat ggaggagaga cct gct cgga cggggagagg aaggccaggt acccggat ga acat ggat ga agat ct t cca gct t cat gca gcagt cccct t ggt gt gt cc act at gacct t ggt ccggca aggagt at gc ggaagt ggaa act acct cac t gcaggat t a gcagccaccc t t gagct gt t cggccgcgag cgt gcgcgt c gt acct gcag ggaat gcaat gaggggggac gccagaagac ct gt accat g gt ct cat cgc cgct gacaac ggagt gcat g t gagaacaga ccct ggagaa ct gcacagac ct t cgacggg ct gcggcagt ct cagt gaaa t gacggggag

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   2018203206 08 May 2018 gt gaat gt ga t acat cat t c t ccgt ggt cc ggcat ccaga t t t gggaact t cat cccct g agaat cct t a ct ggat gt ct t gcgacacca aggacggcca gagt gt gagt gagccact gg aaaat cct gg gt ggct ggcc cact gccaga ggaggcct gg gacat ct cgg agaggcccat t gct gct ggg t gaagcagac acaat gacct cct ggaaagt ccacct gcca ccagt gacgt gcat t t acga t t gct gcct a cat t gt gccc ggcgct at aa cct gccct gt at gagct t t t ggcgt t t t gc t t t gccact g t ggt gcct cc aaccgccgt t gaaggat gag caaagccct c at accaggag caccagcagc gagct cgcag t aacaacat c ct t ccaggac cacct gct cc t gcccacgt g ccagagct gc cagct gt gca gcagt gt gt g gcagacct gc ct caggaaag tgat gttgtc cacagat gcc gcacgat 11 c

   97047_1 act cact 11 g t ccgt ggt ct aaagt gt gt g aacct ccaag t gt gct gaca at gaagcaga t gcaacaagc t gt gagt cca t gt gcccagc gaggagagga cct gcct gt c gagggct gcc gt t gaccct g aaagt cacct aacct cacct ccggt gagcc t act gcagca aggt ggt gga gggaccgcca gcct gt gt gg t ggaggaaga ccagaaaagt cgat ggt gga t ggt ggaccc t t ggggact g at ggcaaggt at ct ccggga aagt cacgt g at gcccact g aagact gt cc t gaat cccag gt gaagcct g ccaccact ct ggct act gga gt ct ggccgg cct gagcat c gaat t t t gat ccct gt ggac gcct ct ggac t t cct cct gt cgagccat at cgcct gct t c ggt gacct gg gaacgggt at t cagcaccct ccct ccaggg agt gt gt gag t gaccct gag ccaggagccg gt at gt ggag cct ggt ct t c

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   2018203206 08 May 2018 ct gct ggat g gt ggacat ga t accacgacg cggcgcat t g t t gaaat aca gccct gct cc gt ccagggcc aacct caagc agcagt gt gg gcccct gaag gggct ct t gg t t cgt cct gg at ggaggagg cagt act cct at cct gcagc gccct gcggt cccaacct gg gct cct ccag t ggagcggct gct cccacgc ccagccaggt cact gt t cca t gat ggccag t gaagaagaa agat ccgcct at gagct gga cccct cct cc gggt t t cgac aaggat cgga t gat t cagcg acat ggt gac gggt gcgaga acct ct ct ga t ct acat ggt gct gt ccgag gcgcat ct cc ct acat cggg gaagt at gcg aat ct t cagc ccaggagccc gaaggt cat t cat cgagaag gcagcaaagg t act ct gccc cct ggggccc caaaat t ggt gat ggat gt g cgt ggagt ac gat ccgct ac ccacagct t c caccggaaat

   97047_1 gctgagtttg cagaagt ggg ct caaggacc ggcagccagg aagat cgacc caacggat gt gt gat cccgg caggcccct g gacgagat cg cccgacat gg aagaggaact gaagccgact ggccaggaca ccct t cagcg cagggcggca t t ggt cagcc cct gcct ct g aagt gct gaa t ccgcgt ggc ggaagcgacc t ggcct ccac gccct gaagc cccggaact t t gggcat t gg agaacaaggc t t agct acct cacaagt cac ccat ggt t ct t caacaggag gcat ccacgt aggcacagt c acaggaccaa agggt gaccg at gagat caa ggcct t t gt g cgt ggt ggag gt cagagct g cagcgaggt c ct cccgcat c t gt ccgct ac gccccat gcc ct t cgt gct g ct gt gacct t t gt gggcccg ggat gt ggcg caaggagt t c cacggt gct g caaaggggac cact gggct g ggagcaggcg gaggct gcct

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   Page 6

   2018203206 08 May 2018 ggagacat cc aggattggct gct cct gacc t cccct gcac agt t t cccag gccaat at ag at t gacgt gc at gcagcggg t t gact t cag acggacgt ct acagt gt t cc ggcccagcag gt cacct t gg gat gaggat g accgt gact t cgggggct ga ggct gccgct aggt ggt gcc ggcccaat gc t ggt gct gca ct gact gcag ct t ct t at t t ggcct cgt ct cat ggaacgt agggaggccc aaat gcat gg ct gt ggat t c ct at t ggaat gcgact ccaa gcaat t cct t ggaat gagaa gccagccaga ggcct t cgt g ggacct gccc cat t ggagt g ccct at cct c gaggt gct gc ccagcccct g t gat gaaat g cact caggt g ggt cccggag cagccaaat c t gccaggccg agt ggat gca t ggagat cgc cgt ggt gaag cct ccacaaa gaggcccggg t ggccagacc ccct aacagc ct gygt gt gc

   97047_1 ggccct aat g at ccaggact t ccggagagg gacgt gat cc aagagt t t cg t cagt gct gc aaagcccat t ggggat gcct ggagcct caa gcagct gat g t acgat gcag ct ccagcgaa ct gt gct ct g gacgt ct gga t t gct gaaga cagt cccct g acaggcagct ccaacgt gca t t gagacgct ggct gcagat t t ct cct gga ccaaggct t t agt at ggaag t gct gagcct tgggctttgc aggcggt ggt ccgccaggt c cccagct acg t cgaagacct gat t t gt t ag cct t gccaga gt cat cgggt t t aaagt gga ccact cggca ggagct ggag cccccgagag ccccaccct c t ggct cct cc cat t t caaaa cat caccacc t gt ggacgt c t gt gcgat ac cat cct ggt c caacagagt g gat ct t ggca ccct accat g gat t t gcat g ccagt gccac caact gt gac agagacct gt cat cgt gacc

   4920

   4980

   5040

   5100

   5160

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   5400

   5460

   5520

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   Page 7

   2018203206 08 May 2018 tttgatgggc gagcaggacc t gcat gaaat gaggt gacgg aacgt t t at g t t cact ccac acgt at ggt c ggcacagt ca cagacgt gcc gt cct cct ct gccat ct gcc gcccacct ct at gt cat gcc gat ggcaacg aaagt cat gt gat ggagt cc t gcacat gcc agaat t t caa t ggaggt gat ccat cgaggt t gaat gggag gt gccat cat aaaacaat ga t gt gt gggat ccacagact g agcccat cct t accact gt t agcaggacag gt cggaccaa caccat ct ct t gagct cct g t ggaaggcag agcaccagt t t cagcgggcg gct gact ggc t ct ccat aat gaagcacagt act ggt ct ct gcat gaggt c gt t ccaact g ct gt gat gag gaaaacact t ggaggagcag t gct gaat gc

   11 gccaccag cggggt ct gc ggt ct acaac t ggggaccat ct gt gt ccct cct ggaagcc gaaggt caac

   97047_1 agct gt t ct t ggt gcct gca gccct ct ccg gt t cct t acg agat t caat c cagct cagcc aacggagcca gt t caggaat t gt ct t gt cc cacaaggt cc gagcaagt gt gt t gact gga cact gt gagc ccct ccgaag gaagaggcct t gggt cccgg t gcacaacgc at gt cct at t gccct ggagc t cgagst gca t gggt gggaa acct t ggt ca ccaagact t t at gact t cat ggact gt gca ccgacagct c t ggct ccagc gt gaggt gat ggacacct ga at ggct gt cc gct gt t t ct g gcact cagt g accaccagcc agccct gccc t caaaacaag aaggcagggc cagt gacat g cat ggaagt c cat ct t caca t gct t caaag gct gagggat gcggccaggg ccact gccag cacat t ct at cgcct ct t at t t t ct gt gct ccggcact gt ccct ccagat cat t ggt gag ct gt cagat c cacggccaaa

   5940

   6000

   6060

   6120

   6180

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   6300

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   6420

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   6540

   6600

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   Page 8

   2018203206 08 May 2018 gct cccacgt cccgagt at g gaacgt ggcc gcct gcagga cccaccct t c t ccacagt ga accacaacca ggccagt t ct at gggcct cc t t cact t acg gt ggt gact g t gggcct ccc t t t at acaac t t t cagct ga gcct gcat gc acgacct gcc aagaccacct gt ggcct gt g agt gt gt gt g t ccagcccac aggaggagt g ggaagaccca gct gt cccct cct gcct t cc gggaggaggg gcgt ggccca t t ct gcat ga gct caccgcg cggagaaccc aaaggaacgt gct gt aagac t caat ggcac gct gcat ggt gcaacccct g t gaagt agcc t gacccagt g act gaccaac caaaagagt g gt gct gt gat tgggtacttg cgacaaggt g ct gcgat gt g gt gct cccag aggcgagt gc gggggact cc ct gcct cat c ct cct gcccc ct cagcgt gc tgtcattggg gcaggtgggg ccccct gggt

   97047_1 cgcct ccgcc agct gt gacc cct ggcgagt t ccccaccct gagt at gagt gcct caaccg t gt gt ccacc t gcacct gca aagccct gt g t gt ggaaggt cagt ct t cct aat gagt gt g cagct ggagg t gcccaagct cccgggaaga gt cat ct ct g t acaaggaag agaat gcaga t gcccccagt gcagacccaa cct gcccccc gt gcct gcaa ccaccaat ga gaagcaccat ccgacat gga aggacagct g gcct gccat c ggaagagt gt t ccgagt gaa t ccct gt ct g gt cgct gt ga ct gt gat gat gat t caagct aaaat aacac ccagt gct gc gcct cact gt ct t cacct gc gcaccgt t t g ct gt gt caac ct gt ggct gt ct accct gt g ggat gccgt g t cggt cgggc t gcct gt gag cggct cccag ggaggaggt c cccct cgggc gcgcat ggag cgat gt gt gc ggagt gcagg aggt gaat gt

   6960

   7020

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   7140

   7200

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   7560

   7620

   7680

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   Page 9

   97047 1

   2018203206 08 May 2018

   t gt gggagat gt t t gcct ac ggct t gcacc at t cagct aa gaggaggaca gat cat gaca 7980 ct gaagcgt g at gagacgct ccaggat ggc t gt gat act c act t ct gcaa ggt caat gag 8040 agaggagagt act t ct ggga gaagagggt c acaggct gcc caccct t t ga t gaacacaag 8100 t gt ct t gct g agggaggt aa aat t at gaaa at t ccaggca cct gct gt ga cacat gt gag 8160 gagcct gagt gcaacgacat cact gccagg ct gcagt at g t caaggt ggg aagct gt aag 8220 t ct gaagt ag aggt ggat at ccact act gc cagggcaaat gt gccagcaa agccat gt ac 8280 t ccat t gaca t caacgat gt gcaggaccag t gct cct gct gct ct ccgac acggacggag 8340 cccat gcagg t ggccct gca ct gcaccaat ggct ctgttg t gt accat ga ggt t ct caat 8400 gccat ggagt gcaaat gct c ccccaggaag t gcagcaagt ga 8442

   <210> 2 <211> 2813 <212> PRT <213> Homo sapi ens <220>

   <221> mi sc_f eat ur e <222> (1)..(22) <223> VWF Si gnal Pept i de <220>

   <221> mi sc_f eat ur e <222> (23)..(763) <223> VWF D1D2 r egi on

   Page 10

   97047 1

   2018203206 08 May 2018 <220>

   <221> m sc_f eat ur e <222> (764)..(866) <223> VWF D Domai n <220>

   <221> m sc_f eat ur e <222> ( 867) . . ( 1240) <223> VWF D3 Domai n <220>

   <221> m sc_f eat ur e <222> ( 1241) . . ( 1479) <223> VWF A1 Domai n <220>

   <221> m sc_f eat ur e <222> ( 2016) . . ( 2016) <223> Xaa can be any naturally <400> 2

   Met Ile Pro Al a Arg Phe Al a Gl y 1 5

   Leu Pro Gl y Thr Leu Cys Al a Gl u 20

   Al a Arg Cys Ser Leu Phe Gl y Ser 35 40 occur r i ng ami no aci d

   Val Leu 10 Leu Al a Leu Al a Leu 15 I l e Gl y Thr Ar g Gl y Ar g Ser Ser Thr 25 30 Asp Phe Val As n Thr Phe As p Gl y 45

   Page 11

   97047 1

   2018203206 08 May 2018

   Ser Met 50 Ty r Ser Phe Al a Gl y Ty r 55 Cy s Ser Ty r Leu 60 Leu Al a Gl y Gl y Cys Gl n Lys Ar g Ser Phe Ser I l e I l e Gl y As p Phe Gl n As n Gl y Lys 65 70 75 80 Arg Val Ser Leu Ser Val Ty r Leu Gly Gl u Phe Phe As p I l e Hi s Leu 85 90 95 Phe Val As n Gl y Thr Val Thr Gl n Gly As p Gl n Ar g Val Ser Met Pr o 100 105 110 Tyr Al a Ser Lys Gl y Leu Ty r Leu Gl u Thr Gl u Al a Gl y Ty r Ty r Lys 115 120 125 Leu Ser Gl y Gl u Al a Ty r Gl y Phe Val Al a Ar g Ile As p Gl y Ser Gl y 130 135 140 Asn Phe Gl n Val Leu Leu Ser As p Ar g Ty r Phe As n Lys Thr Cy s Gl y 145 150 155 160 Leu Cy s Gl y As n Phe As n I l e Phe Al a Gl u As p As p Phe Met Thr Gl n 165 170 175 Gl u Gl y Thr Leu Thr Ser Asp Pr o Ty r As p Phe Al a As n Ser Tr p Al a

   Page 12

   97047 1

   2018203206 08 May 2018

   180 185 190 Leu Ser Ser Gl y Gl u Gl n Tr p Cy s Gl u Ar g Al a Ser Pr o Pr o Ser Ser 195 200 205 Ser Cy s As n I l e Ser Ser Gl y Gl u Met Gl n Lys Gl y Leu Tr p Gl u Gl n 210 215 220 Cys Gl n Leu Leu Lys Ser Thr Ser Val Phe Al a Ar g Cy s Hi s Pr o Leu 225 230 235 240 Val Asp Pr o Gl u Pr o Phe Val Al a Leu Cy s Gl u Lys Thr Leu Cy s Gl u 245 250 255 Cys Al a Gl y Gl y Leu Gl u Cy s Al a Cy s Pr o Al a Leu Leu Gl u Ty r Al a 260 265 270 Arg Thr Cy s Al a Gl n Gl u Gl y Met Val Leu Ty r Gl y Tr p Thr As p Hi s 275 280 285 Ser Al a Cy s Ser Pr o Val Cy s Pr o Al a Gl y Met Gl u Ty r Ar g Gl n Cy s 290 295 300 Val Ser Pr o Cy s Al a Ar g Thr Cy s Gl n Ser Leu Hi s I l e As n Gl u Met 305 310 315 320

   Page 13

   97047 1

   2018203206 08 May 2018

   Cys Gl n Gl u Ar g Cy s 325 Val As p Gl y Cy s Ser 330 Cy s Pr o Gl u Gl y Gl n 335 Leu Leu As p Gl u Gl y Leu Cy s Val Gl u Ser Thr Gl u Cy s Pr o Cy s Val Hi s 340 345 350 Ser Gl y Lys Ar g Ty r Pr o Pr o Gl y Thr Ser Leu Ser Ar g As p Cy s As n 355 360 365 Thr Cy s I l e Cy s Ar g As n Ser Gl n Tr p Ile Cy s Ser As n Gl u Gl u Cy s 370 375 380 Pr o Gl y Gl u Cy s Leu Val Thr Gl y Gl n Ser Hi s Phe Lys Ser Phe As p 385 390 395 400 Asn Ar g Ty r Phe Thr Phe Ser Gl y Ile Cy s Gl n Ty r Leu Leu Al a Ar g 405 410 415 Asp Cy s Gl n As p Hi s Ser Phe Ser Ile Val Ile Gl u Thr Val Gl n Cy s 420 425 430 Al a As p As p Ar g As p Al a Val Cy s Thr Ar g Ser Val Thr Val Ar g Leu 435 440 445 Pr o Gl y Leu Hi s As n Ser Leu Val Lys Leu Lys Hi s Gl y Al a Gl y Val

   Page 14

   2018203206 08 May 2018

   97047_1

   450 455 460

   Al a 465 Met As p Gl y Gl n As p 470 Ile Gl n Leu Pr o Leu 475 Leu Lys Gl y As p Leu 480 Ar g I l e Gl n Hi s Thr Val Thr Al a Ser Val Ar g Leu Ser Ty r Gl y Gl u 485 490 495 Asp Leu Gl n Met As p Tr p As p Gl y Ar g Gl y Ar g Leu Leu Val Lys Leu 500 505 510 Ser Pr o Val Tyr Al a Gl y Lys Thr Cy s Gl y Leu Cy s Gl y As n Ty r As n 515 520 525 Gl y As n Gl n Gl y As p As p Phe Leu Thr Pr o Ser Gl y Leu Al a Gl u Pr o 530 535 540 Ar g Val Gl u As p Phe Gl y As n Al a Tr p Lys Leu Hi s Gl y As p Cy s Gl n 545 550 555 560 As p Leu Gl n Lys Gl n Hi s Ser As p Pr o Cy s Al a Leu As n Pr o Ar g Met 565 570 575 Thr Ar g Phe Ser Gl u Gl u Al a Cys Al a Val Leu Thr Ser Pr o Thr Phe 580 585 590

   Page 15

   2018203206 08 May 2018

   97047_1

   Gl u Al a Cys Hi s Ar g Al a Val Ser 600 Pr o Leu Pr o Tyr Leu 605 Ar g As n Cys 595 Ar g Tyr As p Val Cys Ser Cys Ser As p Gl y Ar g Gl u Cys Leu Cys Gl y 610 615 620 Al a Leu Al a Ser Tyr Al a Al a Al a Cys Al a Gl y Ar g Gl y Val Ar g Val 625 630 635 640 Al a Tr p Ar g Gl u Pr o Gl y Ar g Cys Gl u Leu As n Cys Pr o Lys Gl y Gl n 645 650 655 Val Tyr Leu Gl n Cys Gl y Thr Pr o Cys As n Leu Thr Cys Ar g Ser Leu 660 665 670 Ser Tyr Pr o As p Gl u Gl u Cys As n Gl u Al a Cys Leu Gl u Gl y Cys Phe 675 680 685 Cy s Pr o Pr o Gl y Leu Tyr Met As p Gl u Ar g Gl y As p Cys Val Pr o Lys 690 695 700 Al a Gl n Cys Pr o Cys Tyr Tyr As p Gl y Gl u Ile Phe Gl n Pr o Gl u As p 705 710 715 720 I l e Phe Ser As p Hi s Hi s Thr Met Cys Tyr Cys Gl u As p Gl y Phe Met

   Page 16

   2018203206 08 May 2018

   97047_1

   725 730 735

   Hi s Cys Thr Met 740 Ser Gl y Val Pr o Gl y 745 Ser Leu Leu Pr o As p 750 Al a Val Leu Ser Ser Pr o Leu Ser Hi s Ar g Ser Lys Ar g Ser Leu Ser Cys Ar g 755 760 765 Pr o Pr o Met Val Lys Leu Val Cys Pr o Al a As p As n Leu Ar g Al a Gl u 770 775 780 Gly Leu Gl u Cys Thr Lys Thr Cys Gl n As n Tyr As p Leu Gl u Cys Met 785 790 795 800 Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o Pr o Gl y Met Val Ar g 805 810 815 Hi s Gl u Asn Ar g Cys Val Al a Leu Gl u Ar g Cys Pr o Cys Phe Hi s Gl n 820 825 830 Gl y Lys Gl u Tyr Al a Pr o Gl y Gl u Thr Val Lys I l e Gl y Cys As n Thr 835 840 845 Cys Val Cys Ar g Asp Ar g Lys Tr p As n Cys Thr As p Hi s Val Cys As p 850 855 860

   Page 17

   2018203206 08 May 2018

   97047_1

   Al a 865 Thr Cy s Ser Thr Ile 870 Gl y Met Al a Hi s Ty r 875 Leu Thr Phe As p Gl y 880 Leu Lys Ty r Leu Phe Pr o Gl y Gl u Cy s Gl n Ty r Val Leu Val Gl n As p 885 890 895 Ty r Cy s Gl y Ser As n Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys 900 905 910 Gl y Cy s Ser Hi s Pr o Ser Val Lys Cy s Lys Lys Ar g Val Thr I l e Leu 915 920 925 Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y Gl u Val As n Val Lys 930 935 940 Ar g Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g 945 950 955 960 Ty r I l e I l e Leu Leu Leu Gl y Lys Al a Leu Ser Val Val Tr p As p Ar g 965 970 975 Hi s Leu Ser I l e Ser Val Val Leu Lys Gl n Thr Ty r Gl n Gl u Lys Val 980 985 990

   Cys G y Leu Cys G y Asn Phe Asp G y Ile G n Asn Asn Asp Leu Thr Page 18

   2018203206 08 May 2018

   97047_1

   995 1000 1005

   Ser Ser 1010 As n Leu Gl n Val Gl u 1015 Gl u As p Pr o Val As p 1020 Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cy s Al a As p Thr Ar g Lys Val Pr o 1025 1030 1035 Leu Asp Ser Ser Pr o Al a Thr Cy s Hi s As n As n Ile Met Lys Gl n 1040 1045 1050 Thr Met Val As p Ser Ser Cy s Ar g Ile Leu Thr Ser As p Val Phe 1055 1060 1065 Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu As p Val 1070 1075 1080 Cys I l e Ty r As p Thr Cy s Ser Cy s Gl u Ser Ile Gl y As p Cy s Al a 1085 1090 1095 Cys Phe Cy s As p Thr Ile Al a Al a Ty r Al a Hi s Val Cy s Al a Gl n 1100 1105 1110 Hi s Gl y Lys Val Val Thr Tr p Ar g Thr Al a Thr Leu Cy s Pr o Gl n 1115 1120 1125

   Page 19

   2018203206 08 May 2018

   97047_1

   Ser Cy s 1130 Gl u Gl u Ar g As n Leu 1135 Ar g Gl u As n Gl y Ty r 1140 Gl u Cy s Gl u Tr p Ar g Ty r Asn Ser Cy s Al a Pr o Al a Cy s Gl n Val Thr Cy s Gl n 1145 1150 1155 Hi s Pr o Gl u Pr o Leu Al a Cy s Pr o Val Gl n Cy s Val Gl u Gl y Cy s 1160 1165 1170 Hi s Al a Hi s Cy s Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu Leu Gl n 1175 1180 1185 Thr Cy s Val As p Pr o Gl u As p Cy s Pr o Val Cy s Gl u Val Al a Gl y 1190 1195 1200 Arg Ar g Phe Al a Ser Gl y Lys Lys Val Thr Leu As n Pr o Ser As p 1205 1210 1215 Pr o Gl u Hi s Cy s Gl n I l e Cy s Hi s Cy s As p Val Val As n Leu Thr 1220 1225 1230 Cys Gl u Al a Cy s Gl n Gl u Pr o Gl y Gl y Leu Val Val Pr o Pr o Thr 1235 1240 1245 As p Al a Pr o Val Ser Pr o Thr Thr Leu Ty r Val Gl u As p I l e Ser

   Page 20

   2018203206 08 May 2018

   1250 1255 97047 _1 1260 Gl u Pr o Pr o Leu Hi s As p Phe Tyr Cys Ser Ar g Leu Leu As p Leu 1265 1270 1275 Val Phe Leu Leu Asp Gl y Ser Ser Ar g Leu Ser Gl u Al a Gl u Phe 1280 1285 1290 Gl u Val Leu Lys Al a Phe Val Val As p Met Met Gl u Ar g Leu Ar g 1295 1300 1305 I l e Ser Gl n Lys Tr p Val Ar g Val Al a Val Val Gl u Ty r Hi s As p 1310 1315 1320 Gly Ser Hi s Al a Tyr Ile Gl y Leu Lys As p Ar g Lys Ar g Pr o Ser 1325 1330 1335 Gl u Leu Arg Ar g Ile Al a Ser Gl n Val Lys Ty r Al a Gl y Ser Gl n 1340 1345 1350 Val Al a Ser Thr Ser Gl u Val Leu Lys Ty r Thr Leu Phe Gl n I l e 1355 1360 1365 Phe Ser Lys Ile As p Ar g Pr o Gl u Al a Ser Ar g Ile Al a Leu Leu 1370 1375 1380

   Page 21

   2018203206 08 May 2018

   97047 1

   Leu Met Al a Ser Gl n Gl u Pr o Gl n Ar g Met Ser Ar g As n Phe Val 1385 1390 1395 Arg Tyr Val Gl n Gl y Leu Lys Lys Lys Lys Val Ile Val I l e Pr o 1400 1405 1410 Val Gl y I l e Gl y Pr o Hi s Al a As n Leu Lys Gl n Ile Ar g Leu I l e 1415 1420 1425 Gl u Lys Gl n Al a Pr o Gl u As n Lys Al a Phe Val Leu Ser Ser Val 1430 1435 1440 Asp Gl u Leu Gl u Gl n Gl n Ar g As p Gl u Ile Val Ser Tyr Leu Cys 1445 1450 1455 Asp Leu Al a Pr o Gl u Al a Pr o Pr o Pr o Thr Leu Pr o Pr o As p Met 1460 1465 1470 Al a Gl n Val Thr Val Gl y Pr o Gl y Leu Leu Gl y Val Ser Thr Leu 1475 1480 1485 Gly Pr o Lys Ar g As n Ser Met Val Leu As p Val Al a Phe Val Leu 1490 1495 1500 Gl u Gl y Ser As p Lys Ile Gl y Gl u Al a As p Phe As n Ar g Ser Lys

   Page 22

   2018203206 08 May 2018

   97047_1

   1505 1510 1515

   Gl u Phe Met Gl u Gl u Val Ile Gl n Ar g Met As p Val Gl y Gl n As p 1520 1525 1530 Ser I l e Hi s Val Thr Val Leu Gl n Tyr Ser Tyr Met Val Thr Val 1535 1540 1545 Gl u Tyr Pr o Phe Ser Gl u Al a Gl n Ser Lys Gl y As p I l e Leu Gl n 1550 1555 1560 Ar g Val Ar g Gl u Ile Ar g Tyr Gl n Gl y Gl y As n Ar g Thr As n Thr 1565 1570 1575 Gl y Leu Al a Leu Ar g Tyr Leu Ser As p Hi s Ser Phe Leu Val Ser 1580 1585 1590 Gl n Gl y Asp Ar g Gl u Gl n Al a Pr o As n Leu Val Tyr Met Val Thr 1595 1600 1605 Gl y As n Pr o Al a Ser As p Gl u Ile Lys Ar g Leu Pr o Gl y As p I l e 1610 1615 1620

   Gl n Val

   Val Pro I l e Gl y Val

   Gly Pro Asn Ala Asn

   Val Gl n Gl u

   1625

   1630

   1635

   Page 23

   2018203206 08 May 2018

   97047_1

   Leu Gl u 1640 Ar g I l e Gl y Tr p Pr o 1645 As n Al a Pr o Ile Leu 1650 I l e Gl n As p Phe Gl u Thr Leu Pr o Ar g Gl u Al a Pr o As p Leu Val Leu Gl n Ar g 1655 1660 1665 Cys Cy s Ser Gl y Gl u Gl y Leu Gl n Ile Pr o Thr Leu Ser Pr o Al a 1670 1675 1680 Pr o As p Cy s Ser Gl n Pr o Leu As p Val Ile Leu Leu Leu As p Gl y 1685 1690 1695 Ser Ser Ser Phe Pr o Al a Ser Ty r Phe As p Gl u Met Lys Ser Phe 1700 1705 1710 Al a Lys Al a Phe Ile Ser Lys Al a As n Ile Gl y Pr o Ar g Leu Thr 1715 1720 1725 Gl n Val Ser Val Leu Gl n Ty r Gl y Ser Ile Thr Thr I l e As p Val 1730 1735 1740 Pr o Tr p As n Val Val Pr o Gl u Lys Al a Hi s Leu Leu Ser Leu Val 1745 1750 1755 Asp Val Met Gl n Ar g Gl u Gl y Gl y Pr o Ser Gl n Ile Gl y As p Al a

   Page 24

   2018203206 08 May 2018

   1760 1765 97047 _1 1770 Leu Gl y Phe Al a Val Ar g Ty r Leu Thr Ser Gl u Met Hi s Gl y Al a 1775 1780 1785 Arg Pr o Gl y Al a Ser Lys Al a Val Val Ile Leu Val Thr As p Val 1790 1795 1800 Ser Val As p Ser Val As p Al a Al a Al a As p Al a Al a Ar g Ser As n 1805 1810 1815 Ar g Val Thr Val Phe Pr o Ile Gl y Ile Gl y As p Ar g Ty r As p Al a 1820 1825 1830 Al a Gl n Leu Ar g I l e Leu Al a Gl y Pr o Al a Gl y As p Ser As n Val 1835 1840 1845 Val Lys Leu Gl n Ar g Ile Gl u As p Leu Pr o Thr Met Val Thr Leu 1850 1855 1860 Gly As n Ser Phe Leu Hi s Lys Leu Cy s Ser Gl y Phe Val Ar g I l e 1865 1870 1875 Cys Met As p Gl u As p Gl y As n Gl u Lys Ar g Pr o Gl y As p Val Tr p 1880 1885 1890

   Page 25

   2018203206 08 May 2018

   97047_1

   Thr Leu 1895 Pr o As p Gl n Cy s Hi s 1900 Thr Val Thr Cy s Gl n 1905 Pr o As p Gl y Gl n Thr Leu Leu Lys Ser Hi s Ar g Val As n Cy s As p Ar g Gl y Leu 1910 1915 1920 Ar g Pr o Ser Cy s Pr o As n Ser Gl n Ser Pr o Val Lys Val Gl u Gl u 1925 1930 1935 Thr Cy s Gl y Cy s Ar g Tr p Thr Cy s Pr o Cy s Val Cy s Thr Gl y Ser 1940 1945 1950 Ser Thr Ar g Hi s Ile Val Thr Phe As p Gl y Gl n As n Phe Lys Leu 1955 1960 1965 Thr Gl y Ser Cy s Ser Ty r Val Leu Phe Gl n As n Lys Gl u Gl n As p 1970 1975 1980 Leu Gl u Val Ile Leu Hi s As n Gl y Al a Cy s Ser Pr o Gl y Al a Ar g 1985 1990 1995 Gl n Gl y Cy s Met Lys Ser Ile Gl u Val Lys Hi s Ser Al a Leu Ser 2000 2005 2010 Val Gl u Xaa Hi s Ser As p Met Gl u Val Thr Val As n Gl y Ar g Leu

   Page 26

   2018203206 08 May 2018

   97047_1

   2015 2020 2025

   Val Ser Val Pr o Ty r Val Gl y Gl y As n Met Gl u Val As n Val Ty r 2030 2035 2040 Gl y Al a I l e Met Hi s Gl u Val Ar g Phe As n Hi s Leu Gl y Hi s I l e 2045 2050 2055 Phe Thr Phe Thr Pr o Gl n As n As n Gl u Phe Gl n Leu Gl n Leu Ser 2060 2065 2070 Pr o Lys Thr Phe Al a Ser Lys Thr Ty r Gl y Leu Cy s Gl y I l e Cy s 2075 2080 2085 As p Gl u As n Gl y Al a As n As p Phe Met Leu Ar g As p Gl y Thr Val 2090 2095 2100 Thr Thr As p Tr p Lys Thr Leu Val Gl n Gl u Tr p Thr Val Gl n Ar g 2105 2110 2115 Pr o Gl y Gl n Thr Cy s Gl n Pr o Ile Leu Gl u Gl u Gl n Cy s Leu Val 2120 2125 2130

   Pro Asp

   Ser Ser Hi s Cys G n

   Val

   Leu Leu Leu Pro

   Leu Phe Al a

   2135

   2140

   2145

   Page 27

   2018203206 08 May 2018

   97047 1

   Gl u Cys 2150 Hi s Lys Val Leu Al a 2155 Pr o Al a Thr Phe Tyr 2160 Al a I l e Cys Gl n Gl n Asp Ser Cys Hi s Gl n Gl u Gl n Val Cys Gl u Val I l e Al a 2165 2170 2175 Ser Tyr Al a Hi s Leu Cys Ar g Thr As n Gl y Val Cys Val As p Tr p 2180 2185 2190 Arg Thr Pr o As p Phe Cys Al a Met Ser Cys Pr o Pr o Ser Leu Val 2195 2200 2205 Tyr As n Hi s Cys Gl u Hi s Gl y Cys Pr o Ar g Hi s Cys As p Gl y As n 2210 2215 2220 Val Ser Ser Cys Gl y As p Hi s Pr o Ser Gl u Gl y Cys Phe Cys Pr o 2225 2230 2235 Pr o As p Lys Val Met Leu Gl u Gl y Ser Cys Val Pr o Gl u Gl u Al a 2240 2245 2250 Cys Thr Gl n Cys I l e Gl y Gl u As p Gl y Val Gl n Hi s Gl n Phe Leu 2255 2260 2265 Gl u Al a Tr p Val Pr o As p Hi s Gl n Pr o Cys Gl n Ile Cys Thr Cys

   Page 28

   2018203206 08 May 2018

   2270 2275 97047 _1 2280 Leu Ser Gl y Ar g Lys Val As n Cy s Thr Thr Gl n Pr o Cy s Pr o Thr 2285 2290 2295 Al a Lys Al a Pr o Thr Cy s Gl y Leu Cy s Gl u Val Al a Ar g Leu Ar g 2300 2305 2310 Gl n As n Al a As p Gl n Cy s Cy s Pr o Gl u Ty r Gl u Cy s Val Cy s As p 2315 2320 2325 Pr o Val Ser Cy s As p Leu Pr o Pr o Val Pr o Hi s Cy s Gl u Ar g Gl y 2330 2335 2340 Leu Gl n Pr o Thr Leu Thr As n Pr o Gl y Gl u Cy s Ar g Pr o As n Phe 2345 2350 2355 Thr Cy s Al a Cy s Ar g Lys Gl u Gl u Cy s Lys Ar g Val Ser Pr o Pr o 2360 2365 2370 Ser Cy s Pr o Pr o Hi s Ar g Leu Pr o Thr Leu Ar g Lys Thr Gl n Cy s 2375 2380 2385 Cy s As p Gl u Ty r Gl u Cy s Al a Cy s As n Cy s Val As n Ser Thr Val 2390 2395 2400

   Page 29

   97047_1

   2018203206 08 May 2018

   Ser Cy s 2405 Pr o Leu Gl y Ty r Leu 2410 Al a Ser Thr Al a Thr 2415 As n As p Cy s Gl y Cy s Thr Thr Thr Thr Cy s Leu Pr o As p Lys Val Cy s Val Hi s 2420 2425 2430 Ar g Ser Thr Ile Ty r Pr o Val Gl y Gl n Phe Tr p Gl u Gl u Gl y Cy s 2435 2440 2445 As p Val Cy s Thr Cy s Thr As p Met Gl u As p Al a Val Met Gl y Leu 2450 2455 2460 Ar g Val Al a Gl n Cy s Ser Gl n Lys Pr o Cy s Gl u As p Ser Cy s Ar g 2465 2470 2475 Ser Gl y Phe Thr Ty r Val Leu Hi s Gl u Gl y Gl u Cy s Cy s Gl y Ar g 2480 2485 2490 Cy s Leu Pr o Ser Al a Cy s Gl u Val Val Thr Gl y Ser Pr o Ar g Gl y 2495 2500 2505 As p Ser Gl n Ser Ser Tr p Lys Ser Val Gl y Ser Gl n Tr p Al a Ser 2510 2515 2520 Pr o Gl u As n Pr o Cy s Leu Ile As n Gl u Cy s Val Ar g Val Lys Gl u

   Page 30

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   2525 2530 97047 _1 2535 Gl u Val Phe Ile Gl n Gl n Ar g As n Val Ser Cy s Pr o Gl n Leu Gl u 2540 2545 2550 Val Pr o Val Cy s Pr o Ser Gl y Phe Gl n Leu Ser Cy s Lys Thr Ser 2555 2560 2565 Al a Cy s Cy s Pr o Ser Cy s Ar g Cy s Gl u Ar g Met Gl u Al a Cy s Met 2570 2575 2580 Leu As n Gl y Thr Val Ile Gl y Pr o Gl y Lys Thr Val Met I l e As p 2585 2590 2595 Val Cy s Thr Thr Cy s Ar g Cy s Met Val Gl n Val Gl y Val I l e Ser 2600 2605 2610 Gl y Phe Lys Leu Gl u Cy s Ar g Lys Thr Thr Cy s As n Pr o Cy s Pr o 2615 2620 2625 Leu Gl y Ty r Lys Gl u Gl u As n As n Thr Gl y Gl u Cy s Cy s Gl y Ar g 2630 2635 2640 Cy s Leu Pr o Thr Al a Cy s Thr Ile Gl n Leu Ar g Gl y Gl y Gl n I l e 2645 2650 2655

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   Met Thr 2660 Leu Lys Ar g As p Gl u 2665 Thr Leu Gl n As p Gl y 2670 Cy s As p Thr Hi s Phe Cy s Lys Val As n Gl u Ar g Gl y Gl u Ty r Phe Tr p Gl u Lys 2675 2680 2685 Ar g Val Thr Gl y Cy s Pr o Pr o Phe As p Gl u Hi s Lys Cy s Leu Al a 2690 2695 2700 Gl u Gl y Gl y Lys Ile Met Lys Ile Pr o Gl y Thr Cy s Cy s As p Thr 2705 2710 2715 Cy s Gl u Gl u Pr o Gl u Cy s As n As p Ile Thr Al a Ar g Leu Gl n Ty r 2720 2725 2730 Val Lys Val Gl y Ser Cy s Lys Ser Gl u Val Gl u Val As p I l e Hi s 2735 2740 2745 Ty r Cy s Gl n Gl y Lys Cy s Al a Ser Lys Al a Met Ty r Ser I l e As p 2750 2755 2760 I l e As n As p Val Gl n As p Gl n Cy s Ser Cy s Cy s Ser Pr o Thr Ar g 2765 2770 2775 Thr Gl u Pr o Met Gl n Val Al a Leu Hi s Cy s Thr As n Gl y Ser Val

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   2018203206 08 May 2018

   97047_1

   2780 2785 2790

   Val Tyr Hi s Gl u Val Leu Asn Al a Met Gl u Cys Lys Cys Ser Pro 2795 2800 2805

   Arg Lys Cys Ser Lys 2810 <210> 3 <211> 19 <212> PRT <213> Homo sapi ens <400> 3

   Met Gl n Ile Gl u Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe 1 5 10 15

   Cys Phe Ser

   <210> 4 <211> 2332 <212> PRT <213> Homo sapi ens <400> 4 Al a Thr Ar g Ar g Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr

   Page 33

   97047_1

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   1 5 10 15 Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g Phe Pr o Pr o 20 25 30 Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e Al a Lys Pr o 50 55 60 Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr Ile Gl n Al a Gl u Val 65 70 75 80 Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser Hi s Pr o Val 85 90 95 Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a 100 105 110 Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys Val 115 120 125 Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu Lys Gl u As n 130 135 140

   Page 34

   97047_1

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   Gl y 145 Pr o Met Al a Ser As p 150 Pr o Leu Cy s Leu Thr 155 Ty r Ser Ty r Leu Ser 160 Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e Gl y Al a Leu 165 170 175 Leu Val Cy s Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr Leu 180 185 190 Hi s Lys Phe I l e Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser Tr p 195 200 205 Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a Ser 210 215 220 Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Ty r Val As n Ar g 225 230 235 240 Ser Leu Pr o Gl y Leu Ile Gl y Cy s Hi s Ar g Lys Ser Val Ty r Tr p Hi s 245 250 255 Val I l e Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e Phe Leu Gl u 260 265 270 Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u I l e

   Page 35

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   97047_1

   275 280 285

   Ser Pr o 290 I l e Thr Phe Leu Thr 295 Al a Gl n Thr Leu Leu 300 Met As p Leu Gl y Gl n Phe Leu Leu Phe Cy s Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y Met 305 310 315 320 Gl u Al a Ty r Val Lys Val As p Ser Cy s Pr o Gl u Gl u Pr o Gl n Leu Ar g 325 330 335 Met Lys As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p Leu Thr As p 340 345 350 Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser Phe 355 360 365 I l e Gl n I l e Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s 370 375 380 Ty r I l e Al a Al a Gl u Gl u Gl u As p Tr p As p Ty r Al a Pr o Leu Val Leu 385 390 395 400 Al a Pr o As p As p Ar g Ser Ty r Lys Ser Gl n Ty r Leu As n As n Gl y Pr o 405 410 415

   Page 36

   97047_1

   2018203206 08 May 2018

   Gl n Ar g I l e Gl y 420 Ar g Lys Ty r Lys Lys 425 Val Ar g Phe Met Al a 430 Ty r Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y I l e 435 440 445 Leu Gl y Pr o Leu Leu Ty r Gl y Gl u Val Gl y As p Thr Leu Leu I l e I l e 450 455 460 Phe Lys As n Gl n Al a Ser Ar g Pr o Ty r As n Ile Ty r Pr o Hi s Gl y I l e 465 470 475 480 Thr As p Val Ar g Pr o Leu Ty r Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys 485 490 495 Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u Ile Phe Lys Ty r Lys 500 505 510 Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s 515 520 525 Leu Thr Ar g Ty r Ty r Ser Ser Phe Val As n Met Gl u Ar g As p Leu Al a 530 535 540 Ser Gl y Leu I l e Gl y Pr o Leu Leu Ile Cy s Ty r Lys Gl u Ser Val As p

   Page 37

   2018203206 08 May 2018

   97047_1

   545 550 555 560

   Gl n Ar g Gl y As n Gl n 565 Ile Met Ser As p Lys 570 Ar g As n Val I l e Leu 575 Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u As n I l e Gl n 580 585 590 Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe 595 600 605 Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val Phe As p Ser 610 615 620 Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p Ty r I l e Leu 625 630 635 640 Ser I l e Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y Ty r 645 650 655 Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr Leu Phe Pr o 660 665 670 Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu Tr p 675 680 685

   Page 38

   97047_1

   2018203206 08 May 2018

   I l e Leu 690 Gl y Cys Hi s As n Ser 695 As p Phe Ar g As n Ar g 700 Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cys As p Lys As n Thr Gl y As p Tyr Tyr Gl u 705 710 715 720 As p Ser Tyr Gl u As p Ile Ser Al a Tyr Leu Leu Ser Lys As n As n Al a 725 730 735 I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Ser Ar g Hi s Pr o Ser Thr Ar g 740 745 750 Gl n Lys Gl n Phe As n Al a Thr Thr Ile Pr o Gl u As n As p I l e Gl u Lys 755 760 765 Thr As p Pr o Tr p Phe Al a Hi s Ar g Thr Pr o Met Pr o Lys I l e Gl n As n 770 775 780 Val Ser Ser Ser As p Leu Leu Met Leu Leu Ar g Gl n Ser Pr o Thr Pr o 785 790 795 800 Hi s Gl y Leu Ser Leu Ser As p Leu Gl n Gl u Al a Lys Tyr Gl u Thr Phe 805 810 815 Ser As p As p Pr o Ser Pr o Gl y Al a Ile As p Ser As n As n Ser Leu Ser

   Page 39

   97047_1

   2018203206 08 May 2018

   820 825 830 Gl u Met Thr Hi s Phe Ar g Pr o Gl n Leu Hi s Hi s Ser Gl y As p Met Val 835 840 845 Phe Thr Pr o Gl u Ser Gl y Leu Gl n Leu Ar g Leu As n Gl u Lys Leu Gl y 850 855 860 Thr Thr Al a Al a Thr Gl u Leu Lys Lys Leu As p Phe Lys Val Ser Ser 865 870 875 880 Thr Ser As n As n Leu Ile Ser Thr Ile Pr o Ser As p As n Leu Al a Al a 885 890 895 Gl y Thr As p As n Thr Ser Ser Leu Gl y Pr o Pr o Ser Met Pr o Val Hi s 900 905 910 Tyr As p Ser Gl n Leu As p Thr Thr Leu Phe Gl y Lys Lys Ser Ser Pr o 915 920 925 Leu Thr Gl u Ser Gl y Gl y Pr o Leu Ser Leu Ser Gl u Gl u As n As n As p 930 935 940 Ser Lys Leu Leu Gl u Ser Gl y Leu Met As n Ser Gl n Gl u Ser Ser Tr p 945 950 955 960

   Page 40

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   97047_1

   Gl y Lys Asn Val Ser Ser Thr Gl u Ser Gl y Arg Leu Phe Lys Gl y Lys 965 970 975

   Arg Al a Hi s Gl y Pro Al a Leu Leu Thr Lys Asp Asn Al a Leu Phe Lys 980 985 990

   Val Ser I l e Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Al a 995 1000 1005

   Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu 1010 1015 1020

   Asn Ser Pro Ser Val Trp Gl n Asn I l e Leu Gl u Ser Asp Thr Gl u 1025 1030 1035

   Phe Lys Lys Val Thr Pr o Leu I l e Hi s Asp Ar g Met Leu Met Asp 1040 1045 1050

   Ly s As n Al a Thr Al a Leu Ar g Leu As n Hi s Met Ser As n Ly s Thr 1055 1060 1065

   Thr Ser Ser Lys Asn Met Gl u Met Val Gl n Gl n Lys Lys Gl u Gl y 1070 1075 1080

   Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe Lys

   Page 41

   2018203206 08 May 2018

   1085 1090 97047 _1 1095 Met Leu Phe Leu Pr o Gl u Ser Al a Ar g Tr p Ile Gl n Ar g Thr Hi s 1100 1105 1110 Gl y Lys As n Ser Leu As n Ser Gl y Gl n Gl y Pr o Ser Pr o Lys Gl n 1115 1120 1125 Leu Val Ser Leu Gl y Pr o Gl u Lys Ser Val Gl u Gl y Gl n As n Phe 1130 1135 1140 Leu Ser Gl u Lys As n Lys Val Val Val Gl y Lys Gl y Gl u Phe Thr 1145 1150 1155 Lys As p Val Gl y Leu Lys Gl u Met Val Phe Pr o Ser Ser Ar g As n 1160 1165 1170 Leu Phe Leu Thr As n Leu As p As n Leu Hi s Gl u As n As n Thr Hi s 1175 1180 1185 As n Gl n Gl u Lys Lys Ile Gl n Gl u Gl u Ile Gl u Lys Lys Gl u Thr 1190 1195 1200 Leu I l e Gl n Gl u As n Val Val Leu Pr o Gl n Ile Hi s Thr Val Thr 1205 1210 1215

   Page 42

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   97047_1

   Gl y Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr Arg 1220 1225 1230

   Gl n Asn Val Gl u Gl y Ser Tyr Asp Gl y Al a Tyr Al a Pro Val Leu 1235 1240 1245

   Gl n Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys 1250 1255 1260

   Hi s Thr Al a Hi s Phe Ser Lys Lys Gly Gu Gu Gu Asn Leu Gl u 1265 1270 1275

   Gl y Leu Gl y Asn Gl n Thr Lys Gl n I l e Val Gl u Lys Tyr Al a Cys 1280 1285 1290

   Thr Thr Arg I l e Ser Pro Asn Thr Ser Gl n Gl n Asn Phe Val Thr 1295 1300 1305

   Gl n Ar g Ser Lys Ar g Al a Leu Lys Gl n Phe Ar g Leu Pr o Leu Gl u 1310 1315 1320

   Gl u Thr Gl u Leu Gl u Lys Ar g I l e I l e Val Asp Asp Thr Ser Thr 1325 1330 1335

   Gl n Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr

   Page 43

   2018203206 08 May 2018

   1340 1345 97047 _1 1350 Gl n I l e As p Ty r As n Gl u Lys Gl u Lys Gl y Al a Ile Thr Gl n Ser 1355 1360 1365 Pr o Leu Ser As p Cy s Leu Thr Ar g Ser Hi s Ser Ile Pr o Gl n Al a 1370 1375 1380 As n Ar g Ser Pr o Leu Pr o Ile Al a Lys Val Ser Ser Phe Pr o Ser 1385 1390 1395 I l e Ar g Pr o Ile Ty r Leu Thr Ar g Val Leu Phe Gl n As p As n Ser 1400 1405 1410 Ser Hi s Leu Pr o Al a Al a Ser Ty r Ar g Lys Lys As p Ser Gl y Val 1415 1420 1425 Gl n Gl u Ser Ser Hi s Phe Leu Gl n Gl y Al a Lys Lys As n As n Leu 1430 1435 1440 Ser Leu Al a Ile Leu Thr Leu Gl u Met Thr Gl y As p Gl n Ar g Gl u 1445 1450 1455 Val Gl y Ser Leu Gl y Thr Ser Al a Thr As n Ser Val Thr Ty r Lys 1460 1465 1470

   Page 44

   97047_1

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   Lys Val 1475 Gl u As n Thr Val Leu 1480 Pr o Lys Pr o As p Leu 1485 Pr o Lys Thr Ser Gl y Lys Val Gl u Leu Leu Pr o Lys Val Hi s Ile Ty r Gl n Lys 1490 1495 1500 As p Leu Phe Pr o Thr Gl u Thr Ser As n Gl y Ser Pr o Gl y Hi s Leu 1505 1510 1515 As p Leu Val Gl u Gl y Ser Leu Leu Gl n Gl y Thr Gl u Gl y Al a I l e 1520 1525 1530 Lys Tr p As n Gl u Al a As n Ar g Pr o Gl y Lys Val Pr o Phe Leu Ar g 1535 1540 1545 Val Al a Thr Gl u Ser Ser Al a Lys Thr Pr o Ser Lys Leu Leu As p 1550 1555 1560 Pr o Leu Al a Tr p As p As n Hi s Ty r Gl y Thr Gl n Ile Pr o Lys Gl u 1565 1570 1575 Gl u Tr p Lys Ser Gl n Gl u Lys Ser Pr o Gl u Lys Thr Al a Phe Lys 1580 1585 1590 Lys Lys As p Thr Ile Leu Ser Leu As n Al a Cy s Gl u Ser As n Hi s

   Page 45

   2018203206 08 May 2018

   97047_1

   1595 1600 1605

   Al a I l e Al a Al a Ile As n Gl u Gl y Gl n As n Lys Pr o Gl u I l e Gl u 1610 1615 1620 Val Thr Tr p Al a Lys Gl n Gl y Ar g Thr Gl u Ar g Leu Cy s Ser Gl n 1625 1630 1635 As n Pr o Pr o Val Leu Lys Ar g Hi s Gl n Ar g Gl u Ile Thr Ar g Thr 1640 1645 1650 Thr Leu Gl n Ser As p Gl n Gl u Gl u Ile As p Ty r As p As p Thr I l e 1655 1660 1665 Ser Val Gl u Met Lys Lys Gl u As p Phe As p Ile Ty r As p Gl u As p 1670 1675 1680 Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Ty r 1685 1690 1695 Phe I l e Al a Al a Val Gl u Ar g Leu Tr p As p Ty r Gl y Met Ser Ser 1700 1705 1710

   Ser Pr o

   Hi s Val Leu Arg Asn

   Ar g Al a Gl n Ser Gl y

   Ser Val Pr o

   1715

   1720

   1725

   Page 46

   97047_1

   2018203206 08 May 2018

   Gl n Phe 1730 Lys Lys Val Val Phe 1735 Gl n Gl u Phe Thr As p 1740 Gl y Ser Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu 1745 1750 1755 Leu Gl y Pr o Tyr Ile Ar g Al a Gl u Val Gl u As p As n I l e Met Val 1760 1765 1770 Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser Ser 1775 1780 1785 Leu I l e Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g 1790 1795 1800 Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys 1805 1810 1815 Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys 1820 1825 1830 Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u Lys As p Val Hi s 1835 1840 1845 Ser Gl y Leu Ile Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu

   Page 47

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   97047_1

   1850 1855 1860

   As n Pr o 1865 Al a Hi s Gl y Ar g Gl n 1870 Val Thr Val Gl n Gl u 1875 Phe Al a Leu Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u 1880 1885 1890 As n Met Gl u Ar g As n Cys Ar g Al a Pr o Cys As n Ile Gl n Met Gl u 1895 1900 1905 As p Pr o Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s Al a I l e As n Gl y 1910 1915 1920 Tyr I l e Met As p Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n 1925 1930 1935 Ar g I l e Ar g Tr p Tyr Leu Leu Ser Met Gl y Ser As n Gl u As n I l e 1940 1945 1950 Hi s Ser I l e Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys 1955 1960 1965 Gl u Gl u Tyr Lys Met Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe

   1970 1975 1980

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   97047_1

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   Gl u Thr 1985 Val Gl u Met Leu Pr o 1990 Ser Lys Al a Gl y Ile 1995 Tr p Ar g Val Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu 2000 2005 2010 Phe Leu Val Ty r Ser As n Lys Cy s Gl n Thr Pr o Leu Gl y Met Al a 2015 2020 2025 Ser Gl y Hi s Ile Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Ty r 2030 2035 2040 Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s Ty r Ser Gl y Ser 2045 2050 2055 I l e As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p I l e Lys Val 2060 2065 2070 As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y Ile Lys Thr Gl n Gl y 2075 2080 2085 Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r Ile Ser Gl n Phe I l e I l e 2090 2095 2100 Met Ty r Ser Leu As p Gl y Lys Lys Tr p Gl n Thr Ty r Ar g Gl y As n

   Page 49

   2018203206 08 May 2018

   2105 2110 97047 _1 2115 Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n Val As p Ser Ser 2120 2125 2130 Gl y I l e Lys Hi s As n Ile Phe As n Pr o Pr o Ile Ile Al a Ar g Ty r 2135 2140 2145 I l e Ar g Leu Hi s Pr o Thr Hi s Ty r Ser Ile Ar g Ser Thr Leu Ar g 2150 2155 2160 Met Gl u Leu Met Gl y Cy s As p Leu As n Ser Cy s Ser Met Pr o Leu 2165 2170 2175 Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n Ile Thr Al a Ser 2180 2185 2190 Ser Ty r Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a 2195 2200 2205 Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val 2210 2215 2220 As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met 2225 2230 2235

   Page 50

   97047_1

   2018203206 08 May 2018

   Lys Val 2240 Thr Gl y Val Thr Thr 2245 Gl n Gl y Val Lys Ser 2250 Leu Leu Thr Ser Met Ty r Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y 2255 2260 2265 Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val Phe 2270 2275 2280 Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu As p 2285 2290 2295 Pr o Pr o Leu Leu Thr Ar g Ty r Leu Ar g Ile Hi s Pr o Gl n Ser Tr p 2300 2305 2310

   Val Hi s

   Gl n I l e Al a Leu Ar g

   Met Gl u Val Leu Gl y

   Cy s Gl u Al a

   2315

   2320

   2325

   Gl n Asp Leu Tyr 2330

   <210> 5 <211> 7053 <212> DNA <213> Homo s api ens

   Page 51

   97047_1 <400> 5

   2018203206 08 May 2018

   at gcaaat ag agct ct ccac ct gct t ct 11 ct gt gcct t t t gcgat t ct g ct t t agt gcc 60 accagaagat act acct ggg t gcagt ggaa ct gt cat ggg act at at gca aagt gat ct c 120 ggt gagct gc ct gt ggacgc aagat t t cct cct agagt gc caaaat ct t t t ccat t caac 180 acct cagt cg t gt acaaaaa gact ct gt 11 gt agaat t ca cggat cacct 111 caacat c 240 gct aagccaa ggccaccct g gat gggt ct g ct aggt cct a ccat ccaggc t gaggt t tat 300 gat acagt gg t cat t acact t aagaacat g gct t cccat c ct gt cagt ct t cat gct gt t 360 ggt gt at cct act ggaaagc tt ctgaggga gct gaat at g at gat cagac cagt caaagg 420 gagaaagaag at gat aaagt ct t ccct ggt ggaagccat a cat at gt ct g gcaggt cct g 480 aaagagaat g gt ccaat ggc ct ct gaccca ct gt gcct t a cct act cat a t ct t t ct cat 540 gt ggacct gg t aaaagact t gaat t caggc ct cat t ggag ccct act agt at gt agagaa 600 gggagt ct gg ccaaggaaaa gacacagacc 11 gcacaaat 11 at act act t t t t gct gt a 660 t t t gat gaag ggaaaagt t g gcact cagaa acaaagaact cct t gat gca ggat agggat 720 gct gcat ct g ct cgggcct g gcct aaaat g cacacagt ca at ggt t at gt aaacaggt ct 780 ct gccaggt c t gat t ggat g ccacaggaaa t cagt ct at t ggcat gt gat t ggaat gggc 840 accact cct g aagt gcact c aat at t cct c gaaggt caca cat t t ct t gt gaggaaccat 900 cgccaggcgt cct t ggaaat ct cgccaat a act t t cct t a ct gct caaac act ct t gat g 960 gacct t ggac agt t t ct act gt t t t gt cat at ct ct t ccc accaacat ga t ggcat ggaa 1020

   Page 52

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   gct t at gt ca aagt agacag ct gt ccagag gaaccccaac t acgaat gaa aaat aat gaa 1080 gaagcggaag act at gat ga t gat ct t act gat t ct gaaa t ggat gt ggt caggt t t gat 1140 gat gacaact ct cct t cct t t at ccaaat t cgct cagt t g ccaagaagca t cct aaaact 1200 t gggt acat t acat t gct gc t gaagaggag gact gggact at gct ccct t agt cct cgcc 1260 cccgat gaca gaagt t at aa aagt caat at t t gaacaat g gccct cagcg gat t ggt agg 1320 aagt acaaaa aagt ccgat t t at ggcat ac acagat gaaa cct t t aagac t cgt gaagct 1380 at t cagcat g aat caggaat ct t gggacct t t act t t at g gggaagt t gg agacacact g 1440 t t gat t at at t t aagaat ca agcaagcaga ccat at aaca t ct accct ca cggaat cact 1500 gat gt ccgt c ct t t gt at t c aaggagat t a ccaaaaggt g t aaaacat t t gaaggat t t t 1560 ccaat t ct gc caggagaaat at t caaat at aaat ggacag t gact gt aga agat gggcca 1620 act aaat cag at cct cggt g cct gacccgc t at t act ct a gt t t cgt t aa t at ggagaga 1680 gat ct agct t caggact cat t ggccct ct c ct cat ct gct acaaagaat c t gt agat caa 1740 agaggaaacc agat aat gt c agacaagagg aat gt cat cc t gt t t t ct gt at t t gat gag 1800 aaccgaagct ggt acct cac agagaat at a caacgct 11 c t ccccaat cc agct ggagt g 1860 cagct t gagg at ccagagt t ccaagcct cc aacat cat gc acagcat caa tggct at gt t 1920 t t t gat agt t t gcagt t gt c agt t t gt t t g cat gaggt gg cat act ggt a cat t ct aagc 1980 at t ggagcac agact gact t cct t t ct gt c 11 ct t ct ct g gat at acct t caaacacaaa 2040

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   97047_1

   2018203206 08 May 2018

   at ggt ct at g aagacacact caccct at t c ccat t ct cag gagaaact gt ct t cat gt cg 2100 at ggaaaacc caggt ct at g gat t ct gggg t gccacaact cagact t t cg gaacagaggc 2160 at gaccgcct t act gaaggt 11 ct agt t gt gacaagaaca ct ggt gat t a t t acgaggac 2220 agt t at gaag at at t t cagc at act t gct g agt aaaaaca at gccat t ga accaagaagc 2280 t t ct cccaga at t caagaca ccct agcact aggcaaaagc aat t t aat gc caccacaat t 2340 ccagaaaat g acat agagaa gact gaccct t ggt 11 gcac acagaacacc t at gcct aaa 2400 at acaaaat g t ct cct ct ag tgatttgttg at gct ct t gc gacagagt cc t act ccacat 2460 gggct at cct t at ct gat ct ccaagaagcc aaat at gaga ct t t t t ct ga t gat ccat ca 2520 cct ggagcaa t agacagt aa t aacagcct g t ct gaaat ga cacact t cag gccacagct c 2580 cat cacagt g gggacat ggt at 11 acccct gagt caggcc t ccaat t aag at t aaat gag 2640 aaact gggga caact gcagc aacagagt t g aagaaact t g at t t caaagt t t ct agt aca 2700 t caaat aat c t gat t t caac aat t ccat ca gacaat 11 gg cagcaggt ac t gat aat aca 2760 agt t cct t ag gacccccaag t at gccagt t cat t at gat a gt caat t aga t accact ct a 2820 t t t ggcaaaa agt cat ct cc cct t act gag t ct ggt ggac ct ct gagct t gagt gaagaa 2880 aat aat gat t caaagt t gt t agaat caggt t t aat gaat a gccaagaaag ttcatgggga 2940 aaaaat gt at cgt caacaga gagt ggt agg t t at t t aaag ggaaaagagc t cat ggacct 3000 gct t t gt t ga ct aaagat aa t gcct t at t c aaagt t agca t ct ct t t gt t aaagacaaac 3060

   Page 54

   97047_1

   2018203206 08 May 2018

   aaaact t cca at aat t cagc aact aat aga aagact caca 11 gat ggccc at cat t at t a 3120 at t gagaat a gt ccat cagt ct ggcaaaat at at t agaaa gt gacact ga gt t t aaaaaa 3180 gt gacacct t t gat t cat ga cagaat gct t at ggacaaaa at gct acagc 111 gaggct a 3240 aat cat at gt caaat aaaac t act t cat ca aaaaacat gg aaat ggt cca acagaaaaaa 3300 gagggcccca t t ccaccaga t gcacaaaat ccagat at gt cgt t ct t t aa gat gct at t c 3360 t t gccagaat cagcaaggt g gat acaaagg act cat ggaa agaact ct ct gaact ct ggg 3420 caaggcccca gt ccaaagca at t agt at cc 11 aggaccag aaaaat ct gt ggaaggt cag 3480 aat t t ct t gt ct gagaaaaa caaagt ggt a gt aggaaagg gt gaat t t ac aaaggacgt a 3540 ggact caaag agat ggt t t t t ccaagcagc agaaacct at 11 ct t act aa ct t ggat aat 3600 t t acat gaaa at aat acaca caat caagaa aaaaaaat t c aggaagaaat agaaaagaag 3660 gaaacat t aa t ccaagagaa tgtagttttg cct cagat ac at acagt gac t ggcact aag 3720 aat t t cat ga agaacct t t t ct t act gagc act aggcaaa at gt agaagg 11 cat at gac 3780 ggggcat at g ct ccagt act t caagat 111 aggt cat t aa at gat t caac aaat agaaca 3840 aagaaacaca cagct cat t t ct caaaaaaa ggggaggaag aaaact t gga aggct t ggga 3900 aat caaacca agcaaat t gt agagaaat at gcat gcacca caaggat at c t cct aat aca 3960 agccagcaga at t t t gt cac gcaacgt agt aagagagct t t gaaacaat t cagact ccca 4020 ct agaagaaa cagaact t ga aaaaaggat a at t gt ggat g acacct caac ccagt ggt cc 4080

   Page 55

   97047_1

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   aaaaacat ga aacat t t gac cccgagcacc ct cacacaga t agact acaa t gagaaggag 4140 aaaggggcca t t act cagt c t ccct t at ca gat t gcct t a cgaggagt ca t agcat ccct 4200 caagcaaat a gat ct ccat t acccat t gca aaggt at cat cat t t ccat c t at t agacct 4260 at at at ct ga ccagggt cct at t ccaagac aact ct t ct c at ct t ccagc agcat ct t at 4320 agaaagaaag at t ct ggggt ccaagaaagc agt cat t t ct t acaaggagc caaaaaaaat 4380 aacct t t ct t t agccat t ct aacct t ggag at gact ggt g at caaagaga ggt t ggct cc 4440 ct ggggacaa gt gccacaaa 11 cagt caca t acaagaaag 11 gagaacac t gt t ct cccg 4500 aaaccagact t gcccaaaac at ct ggcaaa gt t gaat t gc 11 ccaaaagt t cacat t t at 4560 cagaaggacc t at t ccct ac ggaaact agc aat gggt ct c ct ggccat ct ggat ct cgt g 4620 gaagggagcc t t ct t caggg aacagaggga gcgat t aagt ggaat gaagc aaacagacct 4680 ggaaaagt t c cct t t ct gag agt agcaaca gaaagct ct g caaagact cc ct ccaagct a 4740 t t ggat cct c t t gct t ggga t aaccact at ggt act caga t accaaaaga agagt ggaaa 4800 t cccaagaga agt caccaga aaaaacagct 111 aagaaaa aggat accat t t t gt ccct g 4860 aacgct t gt g aaagcaat ca t gcaat agca gcaat aaat g agggacaaaa t aagcccgaa 4920 at agaagt ca cct gggcaaa gcaaggt agg act gaaaggc t gt gct ct ca aaacccacca 4980 gt ct t gaaac gccat caacg ggaaat aact cgt act act c 11 cagt caga t caagaggaa 5040 at t gact at g at gat accat at cagt t gaa at gaagaagg aagat t t t ga cat t t at gat 5100

   Page 56

   97047_1

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   gaggat gaaa at cagagccc ccgcagct 11 caaaagaaaa cacgacact a t t t t at t gct 5160 gcagt ggaga ggct ct ggga tt atgggatg agt agct ccc cacat gt t ct aagaaacagg 5220 gct cagagt g gcagt gt ccc t cagt t caag aaagt t gt t t t ccaggaat t t act gat ggc 5280 t cct t t act c agccct t at a ccgt ggagaa ct aaat gaac at t t gggact cct ggggcca 5340 t at at aagag cagaagt t ga agat aat at c at ggt aact t t cagaaat ca ggcct ct cgt 5400 ccct at t cct t ct at t ct ag cct t at t t ct t at gaggaag at cagaggca aggagcagaa 5460 cct agaaaaa act t t gt caa gcct aat gaa accaaaact t act t t t ggaa agt gcaacat 5520 cat at ggcac ccact aaaga t gagt 11 gac t gcaaagcct gggct t at t t ct ct gat gt t 5580 gacct ggaaa aagat gt gca ct caggcct g at t ggacccc ttctggt ctg ccacact aac 5640 acact gaacc ct gct cat gg gagacaagt g acagt acagg aat t t gct ct gt t t t t cacc 5700 at ct t t gat g agaccaaaag ct ggt act t c act gaaaat a t ggaaagaaa ct gcagggct 5760 ccct gcaat a t ccagat gga agat cccact 111 aaagaga at t at cgct t ccat gcaat c 5820 aat ggct aca t aat ggat ac act acct ggc 11 agt aat gg ct caggat ca aaggat t cga 5880 t ggt at ct gc t cagcat ggg cagcaat gaa aacat ccat t ct at t cat t t cagt ggacat 5940 gt gt t cact g t acgaaaaaa agaggagt at aaaat ggcac t gt acaat ct ct at ccaggt 6000 gt t t t t gaga cagt ggaaat gt t accat cc aaagct ggaa t t t ggcgggt ggaat gcct t 6060 at t ggcgagc at ct acat gc t gggat gagc acact 1111 c t ggt gt acag caat aagt gt 6120

   Page 57

   97047_1

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   cagact cccc t gggaat ggc 11 ct ggacac at t agagat t 11 cagat t ac agct t cagga 6180 caat at ggac agt gggcccc aaagct ggcc agact t cat t at t ccggat c aat caat gcc 6240 t ggagcacca aggagccct t 11 ct t ggat c aaggt ggat c t gt t ggcacc aat gat t at t 6300 cacggcat ca agacccaggg t gcccgt cag aagt t ct cca gcct ct acat ct ct cagt t t 6360 at cat cat gt at agt ct t ga t gggaagaag t ggcagact t at cgaggaaa 11 ccact gga 6420 acct t aat gg t ct t ct t t gg caat gt ggat t cat ct ggga t aaaacacaa t at 1111 aac 6480 cct ccaat t a t t gct cgat a cat ccgt 11 g cacccaact c at t at agcat t cgcagcact 6540 ct t cgcat gg agt t gat ggg ct gt gat t t a aat agt t gca gcat gccat t gggaat ggag 6600 agt aaagcaa t at cagat gc acagat t act gct t cat cct act t t accaa t at gt 11 gcc 6660 acct ggt ct c ct t caaaagc t cgact t cac ct ccaaggga ggagt aat gc ct ggagacct 6720 caggt gaat a at ccaaaaga gt ggct gcaa gt ggact t cc agaagacaat gaaagt caca 6780 ggagt aact a ct cagggagt aaaat ct ct g ct t accagca t gt at gt gaa ggagt t cct c 6840 at ct ccagca gt caagat gg ccat cagt gg act ct ct t t t 11 cagaat gg caaagt aaag 6900 gt t t t t cagg gaaat caaga ct cct t caca cct gt ggt ga act ct ct aga cccaccgt t a 6960 ct gact cgct acct t cgaat t cacccccag agt t gggt gc accagat t gc cct gaggat g 7020 gaggt t ct gg gct gcgaggc acaggacct c t ac 7053

   Page 58

   97047_1

   2018203206 08 May 2018 <210> 6 <211> 1438 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> BDDD FVI I I <400> 6 Al a Thr Ar g Ar g Tyr Tyr Leu Gl y 1 5 Met Gl n Ser As p Leu Gl y Gl u Leu 20 Ar g Val Pr o Lys Ser Phe Pr o Phe 35 40 Thr Leu Phe Val Gl u Phe Thr As p 50 55 Ar g Pr o Pr o Tr p Met Gl y Leu Leu 65 70 Tyr As p Thr Val Val Ile Thr Leu 85

   Al a Val 10 Gl u Leu Ser Tr p As p 15 Tyr Pr o Val As p Al a Ar g Phe Pr o Pr o 25 30 As n Thr Ser Val Val Tyr Lys Lys 45 Hi s Leu Phe As n I l e Al a Lys Pr o 60 Gl y Pr o Thr Ile Gl n Al a Gl u Val 75 80 Lys As n Met Al a Ser Hi s Pr o Val 90 95

   Page 59

   2018203206 08 May 2018

   Ser Leu Hi s Al a Val 100 Gl y 97047_1 Gl y Al a Val Ser Ty r 105 Tr p Lys Al a Ser Gl u 110 Gl u Ty r As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys Val 115 120 125 Phe Pr o Gl y Gl y Ser Hi s Thr Ty r Val Tr p Gl n Val Leu Lys Gl u As n 130 135 140 Gl y Pr o Met Al a Ser As p Pr o Leu Cy s Leu Thr Ty r Ser Ty r Leu Ser 145 150 155 160 Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e Gl y Al a Leu 165 170 175 Leu Val Cy s Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr Leu 180 185 190 Hi s Lys Phe I l e Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser Tr p 195 200 205 Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a Ser 210 215 220 Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Ty r Val As n Ar g

   225 230 235 240

   Page 60

   97047_1

   2018203206 08 May 2018

   Ser Leu Pr o Gl y Leu 245 Ile Gl y Cy s Hi s Ar g 250 Lys Ser Val Ty r Tr p 255 Hi s Val I l e Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e Phe Leu Gl u 260 265 270 Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u I l e 275 280 285 Ser Pr o I l e Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met As p Leu Gl y 290 295 300 Gl n Phe Leu Leu Phe Cy s Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y Met 305 310 315 320 Gl u Al a Ty r Val Lys Val As p Ser Cy s Pr o Gl u Gl u Pr o Gl n Leu Ar g 325 330 335 Met Lys As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p Leu Thr As p 340 345 350

   Ser Gl u Met

   Asp Val Val

   Arg Phe Asp Asp Asp Asn Ser

   Pr o Ser Phe

   355

   360

   365

   Page 61

   2018203206 08 May 2018

   I l e Gl n I l e Ar g Ser Val Al a 375 97047_1 Tr p Val Hi s Lys Lys Hi s Pr o Lys 380 Thr 370 Ty r I l e Al a Al a Gl u Gl u Gl u As p Tr p As p Ty r Al a Pr o Leu Val Leu 385 390 395 400 Al a Pr o As p As p Ar g Ser Ty r Lys Ser Gl n Ty r Leu As n As n Gl y Pr o 405 410 415 Gl n Ar g I l e Gl y Ar g Lys Ty r Lys Lys Val Ar g Phe Met Al a Ty r Thr 420 425 430 As p Gl u Thr Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y I l e 435 440 445 Leu Gl y Pr o Leu Leu Ty r Gl y Gl u Val Gl y As p Thr Leu Leu I l e I l e 450 455 460 Phe Lys As n Gl n Al a Ser Ar g Pr o Ty r As n Ile Ty r Pr o Hi s Gl y I l e 465 470 475 480 Thr As p Val Ar g Pr o Leu Ty r Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys 485 490 495 Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u Ile Phe Lys Ty r Lys 500 505 510

   Page 62

   97047_1

   2018203206 08 May 2018

   Tr p Thr Val 515 Thr Val Gl u As p Gl y 520 Pr o Thr Lys Ser As p Pr o Ar g 525 Cy s Leu Thr Ar g Ty r Ty r Ser Ser Phe Val As n Met Gl u Ar g As p Leu Al a 530 535 540 Ser Gl y Leu I l e Gl y Pr o Leu Leu Ile Cy s Ty r Lys Gl u Ser Val As p 545 550 555 560 Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val I l e Leu Phe 565 570 575 Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u As n I l e Gl n 580 585 590 Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe 595 600 605 Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val Phe As p Ser 610 615 620 Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p Ty r I l e Leu 625 630 635 640

   Page 63

   97047_1

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   Ser I l e Gl y Al a Gl n 645 Thr As p Phe Leu Ser 650 Val Phe Phe Ser Gl y 655 Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr Leu Phe Pr o 660 665 670 Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu Tr p 675 680 685 I l e Leu Gl y Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y Met Thr Al a 690 695 700 Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p Ty r Ty r Gl u 705 710 715 720 As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys As n As n Al a 725 730 735 I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Pr o Pr o Val Leu Lys Ar g Hi s 740 745 750 Gl n Ar g Gl u I l e Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u I l e 755 760 765 As p Ty r As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u As p Phe As p

   770 775 780

   Page 64

   97047_1

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   I l e 785 Tyr As p Gl u As p Gl u 790 As n Thr Ar g Hi s Tyr Phe 805 Ile Al a Met Ser Ser Ser 820 Pr o Hi s Val Val Pr o Gl n 835 Phe Lys Lys Val Phe Thr 850 Gl n Pr o Leu Tyr Ar g 855 Leu 865 Gl y Pr o Tyr Ile Ar g 870 Al a Phe Ar g As n Gl n Al a 885 Ser Ar g Ser Tyr Gl u Gl u As p Gl n Ar g

   Ser Pr o Ar g 795 Ser Phe Gl n Lys Lys 800 Val Gl u 810 Ar g Leu Tr p As p Tyr 815 Gl y Ar g 825 As n Ar g Al a Gl n Ser 830 Gl y Ser Phe Gl n Gl u Phe Thr 845 As p Gl y Ser Gl u Leu As n Gl u 860 Hi s Leu Gl y Leu Val Gl u As p 875 As n I l e Met Val Thr 880 Tyr Ser 890 Phe Tyr Ser Ser Leu 895 I l e Gl y 905 Al a Gl u Pr o Ar g Lys 910 As n Phe

   900

   Page 65

   2018203206 08 May 2018

   97047_1

   Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His

   915

   920

   925

   Met Al a Pro Thr Lys Asp Gl u Phe Asp Cys Lys Al a Trp Al a Tyr Phe 930 935 940

   Ser Asp Val Asp Leu Gl u Lys Asp Val Hi s Ser Gl y Leu I l e Gl y Pr o 945 950 955 960

   Leu Leu Val Cy s Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n 965 970 975

   Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe Asp Gl u Thr 980 985 990

   Lys Ser Trp Tyr Phe Thr Gl u Asn Met Gl u Arg Asn Cys Arg Al a Pro 995 1000 1005

   Cys Asn Ile Gl n Met Gl u Asp Pro Thr Phe Lys Gl u Asn Tyr Arg 1010 1015 1020

   Phe Hi s Al a I l e Asn Gl y Tyr I l e Met Asp Thr Leu Pro Gl y Leu 1025 1030 1035

   Val Met Al a Gl n Asp Gl n Arg I l e Arg Trp Tyr Leu Leu Ser Met 1040 1045 1050

   Page 66

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   Gl y Ser 1055 As n Gl u As n Ile Hi s 1060 Ser Ile Hi s Phe Ser 1065 Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Ty r Lys Met Al a Leu Ty r As n 1070 1075 1080 Leu Ty r Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser Lys 1085 1090 1095 Al a Gl y I l e Tr p Ar g Val Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s 1100 1105 1110 Al a Gl y Met Ser Thr Leu Phe Leu Val Ty r Ser As n Lys Cy s Gl n 1115 1120 1125 Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n I l e 1130 1135 1140 Thr Al a Ser Gl y Gl n Ty r Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g 1145 1150 1155 Leu Hi s Ty r Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u Pr o 1160 1165 1170

   Page 67

   97047_1

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   Phe Ser 1175 Tr p Ile Lys Val As p 1180 Leu Leu Al a Pr o Met 1185 I l e I l e Hi s Gl y I l e Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r 1190 1195 1200 I l e Ser Gl n Phe Ile Ile Met Ty r Ser Leu As p Gl y Lys Lys Tr p 1205 1210 1215 Gl n Thr Ty r Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe Phe 1220 1225 1230 Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n Ile Phe As n Pr o 1235 1240 1245 Pr o I l e I l e Al a Ar g Ty r Ile Ar g Leu Hi s Pr o Thr Hi s Ty r Ser 1250 1255 1260 I l e Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y Cy s As p Leu As n 1265 1270 1275 Ser Cy s Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a I l e Ser As p 1280 1285 1290 Al a Gl n I l e Thr Al a Ser Ser Ty r Phe Thr As n Met Phe Al a Thr

   1295 1300 1305

   Page 68

   97047_1

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   Tr p Ser 1310 Pr o Ser Lys Al a Tr p 1325 Ar g Pr o Gl n As p Phe 1340 Gl n Lys Thr Val Lys 1355 Ser Leu Leu Ser Ser 1370 Ser Gl n As p Gl y Lys 1385 Val Lys Val Val Val 1400 As n Ser Leu I l e Hi s 1415 Pr o Gl n Ser

   Al a Ar g 1315 Leu Hi s Leu Val As n 1330 As n Pr o Lys Met Lys 1345 Val Thr Gl y Thr Ser 1360 Met Ty r Val Gl y Hi s 1375 Gl n Tr p Thr Phe Gl n 1390 Gl y As n Gl n As p Pr o 1405 Pr o Leu Leu Tr p Val 1420 Hi s Gl n Ile

   Gl n Gl y 1320 Ar g Ser As n Gl u Tr p 1335 Leu Gl n Val Val Thr 1350 Thr Gl n Gl y Lys Gl u 1365 Phe Leu I l e Leu Phe 1380 Phe Gl n As n As p Ser 1395 Phe Thr Pr o Thr Ar g 1410 Ty r Leu Ar g Al a Leu 1425 Ar g Met Gl u

   Page 69

   2018203206 08 May 2018

   97047_1

   Val Leu G y Cys G u Al a G n Asp Leu Tyr 1430 1435 <210> 7 <211> 4371 <212> DNA <213> Art i f i ci al Sequence <220>

   <223> BDDD FVI I I

   <400> 7 at gcaaat ag agct ct ccac ct gct t ct 11 ct gt gcct t t t gcgat t ct g ct t t agt gcc 60 accagaagat act acct ggg t gcagt ggaa ct gt cat ggg act at at gca aagt gat ct c 120 ggt gagct gc ct gt ggacgc aagat t t cct cct agagt gc caaaat ct t t t ccat t caac 180 acct cagt cg t gt acaaaaa gact ct gt 11 gt agaat t ca cggat cacct 111 caacat c 240 gct aagccaa ggccaccct g gat gggt ct g ct aggt cct a ccat ccaggc tgaggtttat 300 gat acagt gg t cat t acact t aagaacat g gct t cccat c ct gt cagt ct t cat gct gt t 360 ggt gt at cct act ggaaagc tt ctgaggga gct gaat at g at gat cagac cagt caaagg 420 gagaaagaag at gat aaagt ct t ccct ggt ggaagccat a cat at gt ct g gcaggt cct g 480 aaagagaat g gt ccaat ggc ct ct gaccca ct gt gcct t a cct act cat a t ct t t ct cat 540 gt ggacct gg t aaaagact t gaat t caggc ct cat t ggag ccct act agt at gt agagaa 600 gggagt ct gg ccaaggaaaa gacacagacc 11 gcacaaat 11 at act act t t t t gct gt a 660

   Page 70

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   t t t gat gaag ggaaaagt t g gcact cagaa acaaagaact cct t gat gca ggat agggat 720 gct gcat ct g ct cgggcct g gcct aaaat g cacacagt ca at ggt t at gt aaacaggt ct 780 ct gccaggt c t gat t ggat g ccacaggaaa t cagt ct at t ggcat gt gat t ggaat gggc 840 accact cct g aagt gcact c aat at t cct c gaaggt caca cat t t ct t gt gaggaaccat 900 cgccaggcgt cct t ggaaat ct cgccaat a act t t cct t a ct gct caaac act ct t gat g 960 gacct t ggac agt t t ct act gt t t t gt cat at ct ct t ccc accaacat ga t ggcat ggaa 1020 gct t at gt ca aagt agacag ct gt ccagag gaaccccaac t acgaat gaa aaat aat gaa 1080 gaagcggaag act at gat ga t gat ct t act gat t ct gaaa t ggat gt ggt caggt t t gat 1140 gat gacaact ct cct t cct t t at ccaaat t cgct cagt t g ccaagaagca t cct aaaact 1200 t gggt acat t acat t gct gc t gaagaggag gact gggact at gct ccct t agt cct cgcc 1260 cccgat gaca gaagt t at aa aagt caat at t t gaacaat g gccct cagcg gat t ggt agg 1320 aagt acaaaa aagt ccgat t t at ggcat ac acagat gaaa cct t t aagac t cgt gaagct 1380 at t cagcat g aat caggaat ct t gggacct t t act t t at g gggaagt t gg agacacact g 1440 t t gat t at at t t aagaat ca agcaagcaga ccat at aaca t ct accct ca cggaat cact 1500 gat gt ccgt c ct t t gt at t c aaggagat t a ccaaaaggt g t aaaacat t t gaaggat t t t 1560 ccaat t ct gc caggagaaat at t caaat at aaat ggacag t gact gt aga agat gggcca 1620 act aaat cag at cct cggt g cct gacccgc t at t act ct a gt t t cgt t aa t at ggagaga 1680

   Page 71

   97047_1

   2018203206 08 May 2018

   gat ct agct t caggact cat t ggccct ct c ct cat ct gct acaaagaat c t gt agat caa 1740 agaggaaacc agat aat gt c agacaagagg aat gt cat cc t gt 111 ct gt at t t gat gag 1800 aaccgaagct ggt acct cac agagaat at a caacgct 11 c t ccccaat cc agct ggagt g 1860 cagct t gagg at ccagagt t ccaagcct cc aacat cat gc acagcat caa tggct at gt t 1920 t t t gat agt t t gcagt t gt c agt t t gt t t g cat gaggt gg cat act ggt a cat t ct aagc 1980 at t ggagcac agact gact t cct t t ct gt c 11 ct t ct ct g gat at acct t caaacacaaa 2040 at ggt ct at g aagacacact caccct at t c ccat t ct cag gagaaact gt ct t cat gt cg 2100 at ggaaaacc caggt ct at g gat t ct gggg t gccacaact cagact t t cg gaacagaggc 2160 at gaccgcct t act gaaggt 11 ct agt t gt gacaagaaca ct ggt gat t a t t acgaggac 2220 agt t at gaag at at t t cagc at act t gct g agt aaaaaca at gccat t ga accaagaagc 2280 t t ct ct caaa acccaccagt ct t gaaacgc cat caacggg aaat aact cg t act act ct t 2340 cagt cagat c aagaggaaat t gact at gat gat accat at cagt t gaaat gaagaaggaa 2400 gat t t t gaca t t t at gat ga ggat gaaaat cagagccccc gcagct t t ca aaagaaaaca 2460 cgacact at t t t at t gct gc agt ggagagg ct ct gggat t at gggat gag t agct cccca 2520 cat gt t ct aa gaaacagggc t cagagt ggc agt gt ccct c agt t caagaa agt t gt t t t c 2580 caggaat t t a ct gat ggct c ct 11 act cag ccct t at acc gt ggagaact aaat gaacat 2640 t t gggact cc t ggggccat a t at aagagca gaagt t gaag at aat at cat ggt aact t t c 2700

   Page 72

   97047_1

   2018203206 08 May 2018

   agaaat cagg cct ct cgt cc ct at t cct t c t at t ct agcc t t at t t ct t a t gaggaagat 2760 cagaggcaag gagcagaacc t agaaaaaac 111 gt caagc ct aat gaaac caaaact t ac 2820 t t t t ggaaag t gcaacat ca t at ggcaccc act aaagat g agt t t gact g caaagcct gg 2880 gct t at t t ct ct gat gt t ga cct ggaaaaa gat gt gcact caggcct gat t ggacccct t 2940 ct ggt ct gcc acact aacac act gaaccct gct cat ggga gacaagt gac agt acaggaa 3000 t t t gct ct gt t t t t caccat ct t t gat gag accaaaagct ggt act t cac t gaaaat at g 3060 gaaagaaact gcagggct cc ct gcaat at c cagat ggaag at cccact t t t aaagagaat 3120 t at cgct t cc at gcaat caa t ggct acat a at ggat acac t acct ggct t agt aat ggct 3180 caggat caaa ggat t cgat g gt at ct gct c agcat gggca gcaat gaaaa cat ccat t ct 3240 at t cat t t ca gt ggacat gt gt t cact gt a cgaaaaaaag aggagt at aa aat ggcact g 3300 t acaat ct ct at ccaggt gt 1111 gagaca gt ggaaat gt t accat ccaa agct ggaat t 3360 t ggcgggt gg aat gcct t at t ggcgagcat ct acat gct g ggat gagcac act t t t t ct g 3420 gt gt acagca at aagt gt ca gact cccct g ggaat ggct t ct ggacacat t agagat t t t 3480 cagat t acag ct t caggaca at at ggacag t gggccccaa agct ggccag act t cat t at 3540 t ccggat caa t caat gcct g gagcaccaag gagccct t t t ct t ggat caa ggt ggat ct g 3600 t t ggcaccaa t gat t at t ca cggcat caag acccagggt g cccgt cagaa gt t ct ccagc 3660 ct ct acat ct ct cagt t t at cat cat gt at agt ct t gat g ggaagaagt g gcagact t at 3720

   Page 73

   97047_1

   2018203206 08 May 2018

   cgaggaaat t ccact ggaac ct t aat ggt c t t ct t t ggca at gt ggat t c at ct gggat a 3780 aaacacaat a t t t t t aaccc t ccaat t at t gct cgat aca t ccgt t t gca cccaact cat 3840 t at agcat t c gcagcact ct t cgcat ggag tt gat gggct gt gat t t aaa t agt t gcagc 3900 at gccat t gg gaat ggagag t aaagcaat a t cagat gcac agat t act gc t t cat cct ac 3960 t t t accaat a t gt t t gccac ct ggt ct cct t caaaagct c gact t cacct ccaagggagg 4020 agt aat gcct ggagacct ca ggt gaat aat ccaaaagagt ggct gcaagt ggact t ccag 4080 aagacaat ga aagt cacagg agt aact act cagggagt aa aat ct ct gct t accagcat g 4140 t at gt gaagg agt t cct cat ct ccagcagt caagat ggcc at cagt ggac t ct ct 11111 4200 cagaat ggca aagt aaaggt 1111 caggga aat caagact cct t cacacc t gt ggt gaac 4260 t ct ct agacc caccgt t act gact cgct ac ct t cgaat t c acccccagag ttgggtgcac 4320 cagat t gccc t gaggat gga ggt t ct gggc t gcgaggcac aggacct ct a c 4371

   <210> 8 <211> 25 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 8

   Page 74

   2018203206 08 May 2018

   97047_1

   Thr Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pro Asn Asp Lys Tyr 1 5 10 15

   Gl u Pr o Phe Tr p Gl u Asp Gl u Gl u Lys 20 25 <210> 9 <211> 4 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 9

   Ar g Ar g Ar g Ar g <210> 10 <211> 6 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 10

   Ar g Lys Ar g Ar g Lys Ar g

   1 5

   Page 75

   97047_1

   2018203206 08 May 2018

   <210> 11 <211> 5 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 11 Ar g Ar g Ar g Ar g Ser 1 5

   <210> 12 <211> 9 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 12 Thr Gl n Ser Phe As n As p Phe Thr Ar g 1 5

   <210>

   <211>

   <212>

   <213>

   PRT

   Art i f i ci al Sequence

   Page 76

   97047_1

   2018203206 08 May 2018 <220>

   <223> cl eavage si t e <400> 13

   Ser Val Ser Gl n Thr Ser Lys Leu Thr Ar g 1 5 10 <210> 14 <211> 10 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 14

   Asp Phe Leu Al a Gl u Gl y Gl y Gl y Val Ar g 1 5 10 <210> 15 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 15

   Page 77

   97047_1

   2018203206 08 May 2018

   Thr Thr 1 Lys I l e Lys Pr o Ar g 5 <210> 16 <211> 5 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 16 Leu Val Pr o Ar g Gl y 1 5 <210> 17 <211> 5 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 17 Al a Leu Ar g Pr o Ar g 1 5 <210> 18 <211> 7

   Page 78

   2018203206 08 May 2018

   97047_1 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 18

   Lys Leu Thr Ar g Al a Gl u Thr

   1 5 <210> 19 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 19

   Asp Phe Thr Ar g Val Val Gl y

   1 5 <210> 20 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e

   Page 79

   2018203206 08 May 2018

   97047_1 <400> 20

   Thr Met Thr Arg Ile Val Gly Gly 1 5 <210> 21 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 21

   Ser Pr o Phe Ar g Ser Thr Gl y Gl y 1 5 <210> 22 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 22

   Leu Gl n Val Ar g I l e Val Gl y Gl y 1 5

   Page 80

   2018203206 08 May 2018

   97047_1 <210> 23 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 23

   Pr o Leu Gl y Ar g I l e Val Gl y Gl y

   1 5 <210> 24 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 24

   I l e Gl u Gl y Ar g Thr Val Gl y Gl y

   1 5 <210> 25 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   Page 81

   97047_1

   2018203206 08 May 2018

   <223> cl eavage si t e <400> 25 Leu Thr Pr o Ar g Ser Leu Leu Val 1 5 <210> 26 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 26 Leu Gl y Pr o Val Ser Gl y Val Pr o 1 5 <210> 27 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 27 Val Al a Gl y As p Ser Leu Gl u Gl u 1 5

   Page 82

   97047_1

   2018203206 08 May 2018

   <210> 28 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 28 Gl y Pr o Al a Gl y Leu Gl y Gl y Al a 1 5

   <210> 29 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 29 Gl y Pr o Al a Gl y Leu Ar g Gl y Al a 1 5

   <210> 30 <211> 8 <212> PRT <213> Ar t i f i c i al Sequenc e

   Page 83

   97047_1

   2018203206 08 May 2018 <220>

   <223> cl eavage si t e <400> 30

   Al a Pr o Leu Gl y Leu Ar g Leu Ar g 1 5 <210> 31 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 31

   Pr o Al a Leu Pr o Leu Val Al a Gl n

   1 5 <210> 32 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 32

   Page 84

   97047_1

   2018203206 08 May 2018

   Gl u Asn Leu Tyr Phe Gl n Gl y 1 5 <210> 33 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 33 Asp Asp Asp Lys I l e Val Gl y Gl y 1 5 <210> 34 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 34 Leu Gl u Val Leu Phe Gl n Gl y Pr o 1 5 <210> 35 <211> 8

   Page 85

   2018203206 08 May 2018

   97047_1 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 35

   Leu Pr o Lys Thr Gl y Ser Gl u Ser

   1 5 <210> 36 <211> 42 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE42 <400> 36

   Gl y Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y

   1 5 10 15

   Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a

   20 25 30

   Thr Ser Gl y Ser

   Gl u Thr

   Pr o Al a

   Ser

   Ser

   Page 86

   97047_1

   2018203206 08 May 2018 <210> 37 <211> 143 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE144 <400> 37

   Gl y Ser Gl u Pr o Al a Thr Ser Gl y 1 5 Ser Al a Thr Pr o Gl u Ser Gl y Pr o 20 Ser Gl u Thr Pr o Gl ^y Ser Pr o Al a 35 40 Gl y Thr Ser Thr Gl u Pr o Ser Gl u 50 55 Al a Thr Ser Gl y Se r Gl u Thr Pr o 65 70 Ser Gl u Thr Pr o Gl ^y Ser Gl u Pr o 85

   Se r Gl u Thr Pr o Gl y Thr Ser Gl u 10 15 Gl ^y Ser Gl u Pr o Al a Thr Ser Gl y 25 30 Gl ^y Ser Pr o Thr Ser Thr Gl u Gl u 45 Gl ^y Ser Al a Pr o Gl y Ser Gl u Pr o 60 Gl ^y Ser Gl u Pr o Al a Thr Ser Gl y 75 80 Al a Thr Ser Gl y Ser Gl u Thr Pr o 90 95

   Page 87

   2018203206 08 May 2018

   Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y 97047_1 Ser Al a Pr o Gl y Thr Ser Gl u 100 105 110 Ser Al a Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser 115 120 125 Gl u Thr Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 130 135 140 <210> 38 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220> <223> AG144 <400> 38 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 1 5 10 15 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr 20 25 30 Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o 35 40 45

   Page 88

   2018203206 08 May 2018

   Gl y Al a Ser Pr o Gl ^y Thr Ser Ser 50 55 Gl y Thr Ser Ser Th r Gl y Ser Pr o 65 70 Thr Gl y Ser Pr o Gl ^y Ser Ser Pr o 85 Gl y Al a Ser Pr o Gl ^y Thr Ser Ser 100 Ser Al a Ser Thr Gl ^y Thr Gl y Pr o 115 120 Ser Ser Ser Pr o Gl ^y Ser Ser Thr 130 135

   Thr 97047_1 Ser Pr o Gl y Ser Pr o 60 Gl y Al a Gl y Ser Ser Thr Pr o Ser Gl y Al a 75 80 Ser Al a Ser Thr Gl y Thr Gl y Pr o 90 95 Thr Gl y Ser Pr o Gl y Ser Ser Pr o 105 110 Gl y Thr Pr o Gl y Ser Gl y Thr Al a 125 Pr o Ser Gl y Al a Thr Gl y Ser Pr o 140

   <210> 39 <211> 288 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE288 <400> 39

   Page 89

   2018203206 08 May 2018

   Gl y 1 Thr Ser Gl u Ser 5 Al a Thr 97047_1 Ser Gl u 15 Pr o Pr o Gl u Ser 10 Gl y Pr o Gl y Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 20 25 30 Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 35 40 45 Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr 50 55 60 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 65 70 75 80 Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 85 90 95 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u 100 105 110 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 115 120 125

   Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u

   130

   135

   140

   Page 90

   97047_1

   2018203206 08 May 2018

   Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u 145 150 155 160 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 165 170 175 Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 180 185 190 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o 195 200 205 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 210 215 220 Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 225 230 235 240 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o 245 250 255

   Al a Thr Ser Gl y Ser

   Gl u Thr

   Pr o

   260

   Gl y Thr Ser 265

   Gl u Ser Al a Thr Pr o

   270

   Page 91

   2018203206 08 May 2018

   97047_1

   Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 275 280 285 <210> 40 <211> 288 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AG288 <400> 40

   Pr o 1 Gl y Al a Ser Pr o 5 Gl y Thr Ser Ser Thr 10 Gl y Ser Pr o Gl y Al a 15 Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr 20 25 30 Al a Ser Ser Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser 35 40 45 Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser 50 55 60 Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr 65 70 75 80

   Page 92

   97047_1

   2018203206 08 May 2018

   Al a Ser Ser Ser Pr o 85 Gl y Ser Ser Thr Pr o 90 Ser Gl y Al a Thr Gl y 95 Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser 100 105 110 Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser 115 120 125 Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 130 135 140 Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser 145 150 155 160 Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser 165 170 175 Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y 180 185 190 Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser 195 200 205 Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y

   210 215 220

   Page 93

   97047_1

   2018203206 08 May 2018

   Al a Thr Gl y Ser Pr o Gl y 225 230

   Pr o Gl y Al a Ser Pr o Gl y 245

   Pr o Ser Al a Ser Thr Gl y 260

   Al a Ser Ser Ser Pr o Gl y 275

   Ser Ser Pr o Ser Al a 235 Thr Ser Ser Thr 250 Gl y Thr Gl y Pr o 265 Gl y Thr Ser Ser 280 Thr Pr o Ser

   Ser Thr Gl y Thr Gl y 240 Ser Pr o Gl y Ser 255 Ser Pr o Gl y Ser 270 Gl y Thr Gl y Al a 285 Thr Gl y Ser

   <210> 41 <211> 576 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE576 <400> 41

   Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u 1 5 10 15

   Ser Al a Thr

   Pr o Gl u Ser Gl y

   Pr o Gl y Thr

   Ser Thr

   Gl u Pr o Ser

   Gl u

   Page 94

   97047_1

   2018203206 08 May 2018

   Gl y

   Gl y

   Gl u

   Gl u

   Gl y

   Gl y

   Gl u

   Gl y 145

   Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 35 40 45 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 50 55 60 Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 70 75 80 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 85 90 95 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a 100 105 110 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o 115 120 125 Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 130 135 140 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a 150 155 160

   Page 95

   2018203206 08 May 2018

   Gl y

   Gl y

   Gl y

   Gl u

   Gl u

   225

   Gl y

   Gl u

   Gl u

   970 47_1 Ser Pr o Thr Ser Thr Gl u Gl u Gl ^y Thr Ser Thr Gl u Pr o Ser Gl u 165 170 175 Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 180 18 5 190 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr 195 200 205 Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 210 215 220 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 230 235 240 Thr Ser Thr Gl u Pr o Ser Gl u Gl ^y Ser Al a Pr o Gl y Thr Ser Thr 245 250 255 Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 260 26 5 270 Ser Gl y Pr o Gl y Thr Ser Gl u Se r Al a Thr Pr o Gl u Ser Gl y Pr o 275 280 285

   290

   295

   300

   Gl y

   Ser Pr o Al a Gl y Ser

   Pr o Thr Ser Thr

   Gl u Gl u

   Gl y Thr Ser Gl u

   Page 96

   97047_1

   2018203206 08 May 2018

   Ser Al a Thr Pr o Gl u

   305

   Ser Gl u Thr Pr o Gl y 325

   Gl y Thr Ser Thr Gl u 340

   Gl u Pr o Ser Gl u Gl y 355

   Gl y Ser Al a Pr o Gl y 370

   Gl y Thr Ser Thr Gl u 385

   Gl u Pr o Ser Gl u Gl y 405

   Ser Thr Gl u Gl u Gl y 420

   Ser 310 Gl y Pr o Gl y Ser Gl u 315 Thr Ser Gl u Ser Al a 330 Thr Pr o Ser Gl u Gl y 345 Ser Al a Ser Al a Pr o 360 Gl y Thr Ser Thr Ser 375 Thr Gl u Pr o Ser Pr o 390 Ser Gl u Gl y Ser Al a 395 Ser Al a Pr o Gl y Ser 410 Pr o Thr Ser Thr Gl u 425 Pr o Ser

   Pr o Al a Thr Ser Gl y 320

   Pr o Gl u Ser Gl y Pr o 335

   Pr o Gl y Thr Ser Thr 350

   Thr Gl u Pr o Ser Gl u

   365

   Gl u Gl y Ser Al a Pr o 380

   Pr o Gl y Thr Ser Thr 400

   Al a Gl y Ser Pr o Thr 415

   Gl u Gl y Ser Al a Pr o

   430

   Page 97

   2018203206 08 May 2018

   Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 97047_1 Ser Gl y Pr o Gl y Ser Gl u Pr o 435 440 445 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 450 455 460 Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 465 470 475 480 Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr 485 490 495 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 500 505 510 Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 515 520 525 Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a 530 535 540 Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o 545 550 555 560

   565

   570

   575

   Gl u Ser

   Gl y Pr o Gl y Thr

   Ser Thr

   Gl u Pr o Ser

   Gl u Gl y Ser Al a Pr o

   Page 98

   97047_1

   2018203206 08 May 2018 <210> 42 <211> 576 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AG576 <400> 42

   Pr o 1 Gl y Thr Pr o Gl y 5 Ser Gl y Thr Al a Ser 10 Ser Ser Pr o Gl y Ser 15 Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser 20 25 30 Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y 35 40 45 Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser 50 55 60 Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser 65 70 75 80 Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 85 90 95

   Page 99

   97047_1

   2018203206 08 May 2018

   Pr o Gl y Al a Ser Pr o Gl ^y Thr Ser Ser Thr Gl y Ser Pr o Gl y Thr Pr o 100 105 110 Gl y Ser Gl y Thr Al a Se r Ser Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser 115 120 125 Ser Thr Gl y Ser Pr o Gl ^y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 130 135 140 Pr o Gl y Al a Ser Pr o Gl ^y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser 145 15 0 155 160 Pr o Ser Al a Ser Thr Gl ^y Thr Gl y Pr o Gl y Thr Pr o Gl y Ser Gl y Thr 165 170 175 Al a Ser Ser Ser Pr o Gl ^y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 180 185 190 Pr o Gl y Al a Ser Pr o Gl ^y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser 195 200 205

   Pr o Gl y Thr

   Ser Ser Thr

   Gl y Ser

   Pr o Gl y Ser Ser Thr

   Pr o Ser Gl y

   210

   215

   220

   Page 100

   97047_1

   2018203206 08 May 2018

   Al a 225 Thr Gl y Ser Pr o Gl y 230 Ser Ser Thr Pr o Ser 235 Gl y Al a Thr Gl y Ser 240 Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Thr Pr o 245 250 255 Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y 260 265 270 Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser 275 280 285 Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser 290 295 300 Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser 305 310 315 320 Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 325 330 335 Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser 340 345 350 Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser

   355 360 365

   Page 101

   97047_1

   2018203206 08 May 2018

   Ser Thr Gl ^y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 370 375 380 Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Thr Pr o 385 390 395 400 Gl y Ser Gl ^y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y 405 410 415 Al a Thr Gl ^y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser 420 425 430 Pr o Gl y Se r Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Thr Pr o 43 5 440 445 Gl y Ser Gl ^y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y 450 455 460 Al a Thr Gl ^y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser 465 470 475 480 Pr o Gl y Se r Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser 485 490 495

   Page 102

   97047_1

   2018203206 08 May 2018

   Pr o Ser Al a Ser Thr Gl ^y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser 500 505 510 Ser Thr Gl y Ser Pr o Gl ^y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser 515 520 525 Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser 530 535 540 Pr o Ser Al a Ser Thr Gl ^y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser 545 55 0 555 560 Thr Gl y Thr Gl y Pr o Gl ^y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser 565 570 575

   <210> 43 <211> 864 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE864 <400> 43

   Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u 1 5 10 15

   Page 103

   2018203206 08 May 2018

   Ser Al a Thr Pr o 20 97047_1 Gl u Ser Gl y Pr o Gl y 25 Thr Ser Thr Gl u Pr o 30 Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 35 40 45 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 50 55 60 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 65 70 75 80 Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 85 90 95 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a 100 105 110 Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o 115 120 125 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 130 135 140 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a 145 150 155 160

   Page 104

   97047_1

   2018203206 08 May 2018

   Gl y

   Gl y

   Gl y

   Gl u

   Gl u

   225

   Gl y

   Gl u

   Gl u

   Ser Pr o Thr Ser Thr Gl u Gl u Gl ^y Thr Ser Thr Gl u Pr o Ser Gl u 165 170 175 Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 180 18 5 190 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr 195 200 205 Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 210 215 220 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 230 235 240 Thr Ser Thr Gl u Pr o Ser Gl u Gl ^y Ser Al a Pr o Gl y Thr Ser Thr 245 250 255 Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 260 26 5 270 Ser Gl y Pr o Gl y Thr Ser Gl u Se r Al a Thr Pr o Gl u Ser Gl y Pr o 275 280 285

   Page 105

   2018203206 08 May 2018

   97047_1

   Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u 290 295 300

   Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y 305 310 315 320

   Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 325 330 335

   Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 340 345 350

   Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u 355 360 365

   Gl y Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 370 375 380

   Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 385 390 395 400

   Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 405 410 415

   Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 420 425 430

   Page 106

   97047_1

   2018203206 08 May 2018

   Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 435 440 445 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 450 455 460 Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 46 5 470 475 480 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr 485 490 495 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 500 50 5 510 Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl ^y Ser Pr o Thr Ser Thr Gl u Gl u 515 520 525 Gl ^y Ser Pr o Al a Gl y Ser Pr o Thr Se r Thr Gl u Gl u Gl y Ser Pr o Al a 530 535 540 Gl ^y Ser Pr o Thr Ser Thr Gl u Gl u Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 54 5 550 555 560

   Page 107

   2018203206 08 May 2018

   Gl u

   Gl y

   Al a

   Gl u

   Gl y 625

   Gl u

   Ser

   Gl y

   Ser Gl y Pr o Gl y Thr Ser Thr Gl u 97047_1 Pr o Ser Gl u Gl y Ser Al a Pr o 565 570 575 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 580 585 590 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 595 600 605 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 610 615 620 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr 630 635 640 Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 645 650 655 Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 660 665 670 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u 675 680 685

   Ser

   Al a Thr

   Pr o Gl u Ser

   Gl y Pr o Gl y Ser

   Pr o Al a Gl y Ser

   Pr o Thr

   690

   695

   700

   Page 108

   97047_1

   2018203206 08 May 2018

   Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 705 710 715 720 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u 725 730 735 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 740 745 750 Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 755 760 765 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o 770 775 780 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 785 790 795 800 Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 805 810 815

   Gl y Thr Ser Thr

   Gl u Pr o Ser

   Gl u

   820

   Gl y Ser Al a Pr o 825

   Gl y Ser Gl u Pr o 830

   Page 109

   2018203206 08 May 2018

   97047_1

   Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o

   835

   840

   845

   Gl u Ser Gl y Pr o Gl y Thr Ser Thr

   Gl u Pr o Ser

   850

   855

   Gl u Gl y Ser Al a Pr o 860 <210> 44 <211> 864 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AG864 <400> 44

   Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Pr o 1 5 10 15

   Ser Al a Ser Thr

   Gl y Thr

   Gl y Pr o Gl y Ser Ser

   Pr o Ser Al a Ser

   Thr

   Gl y Thr

   Gl y Pr o Gl y Thr

   Pr o Gl y Ser

   Gl y Thr Al a Ser

   Ser

   Ser

   Pr o

   Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o

   Page 110

   2018203206 08 May 2018

   Ser 65 Al a Ser Thr Gl y 97047_1 Ser 80 Thr 70 Gl y Pr o Gl y Al a Ser 75 Pr o Gl y Thr Ser Thr Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o 85 90 95 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Thr Pr o Gl y 100 105 110 Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 115 120 125 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 130 135 140 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 145 150 155 160 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 165 170 175 Thr Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o 180 185 190 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o

   195 200 205

   Page 111

   2018203206 08 May 2018

   97047 1

   Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr 210 215 220 Gl y Thr Gl y Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl ^y Ser Pr o 225 230 235 240 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Al a Ser Pr o 245 250 255 Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Th r Ser Ser 260 265 27 0 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl ^y Ser Pr o 275 280 285 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o 290 295 300 Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Th r Ser Ser 305 310 315 320 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl ^y Ser Pr o 325 330 335

   Page 112

   2018203206 08 May 2018

   Gl y Ser Ser Pr o 340 Ser Al a Ser Thr 97047_1 Gl y Thr 350 Pr o Gl y Gl y 345 Thr Gl y Pr o Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 355 360 365 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 370 375 380 Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Thr 385 390 395 400 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 405 410 415 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 420 425 430 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 435 440 445 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 450 455 460 Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o

   465 470 475 480

   Page 113

   97047_1

   2018203206 08 May 2018

   Gl ^y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o 485 490 495 Gl ^y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 500 505 510 Th r Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o 515 520 525 Gl ^y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o 530 535 540 Gl ^y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 54 5 550 555 560 Th r Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 565 570 575 Gl ^y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 580 585 590

   Pr o Ser

   Gl y Al a Thr Gl y Ser

   Pr o Gl y Thr

   Pr o Gl y Ser

   Gl y Thr Al a

   595

   600

   605

   Page 114

   2018203206 08 May 2018

   97047_1

   Ser Ser Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o 610 615 620

   Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 625 630 635 640

   Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 645 650 655

   Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o 660 665 670

   Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o 675 680 685

   Gl y Thr Ser Ser Thr

   Gl y Ser

   Pr o Gl y Thr

   Pr o Gl y Ser

   Gl y Thr Al a

   690

   695

   700

   Ser Ser Ser

   Pr o Gl y Ser

   Ser Thr

   Pr o Ser Gl y Al a Thr

   Gl y Ser

   Pr o

   705

   710

   715

   720

   Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o

   725

   730

   735

   Ser Al a Ser Thr

   Gl y Thr

   Gl y Pr o Gl y Al a Ser

   Pr o Gl y Thr

   Ser

   Ser

   740

   745

   750

   Page 115

   97047_1

   2018203206 08 May 2018

   Thr Gl y Ser 755 Pr o Gl y Al a Ser Pr o 760 Gl y Thr Ser Ser Thr 765 Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o 770 775 780 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 785 790 795 800 Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o 805 810 815 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 820 825 830 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 835 840 845 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 850 855 860

   <210> 45 <211> 9 <212> PRT <213> Ar t i f i c i al Sequenc e

   Page 116

   97047_1

   2018203206 08 May 2018

   <220> <223> cl eavage si t e <400> 45 Thr Gl n Ser Phe As n As p Phe Thr Ar g 1 5 <210> 46 <211> 10 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 46 Ser Val Ser Gl n Thr Ser Lys Leu Thr Ar g 1 5 10 <210> 47 <211> 10 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 47

   Page 117

   97047_1

   2018203206 08 May 2018

   As p Phe 1 Leu Al a Gl u Gl y Gl y Gl y Val Ar g 10 5 <210> 48 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 48 Thr Thr Ly s I l e Ly s Pr o Ar g 1 5 <210> 49 <211> 5 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 49 Leu Val Pr o Ar g Gl y 1 5 <210> 50 <211> 5

   Page 118

   2018203206 08 May 2018

   97047_1 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <400> 50

   Al a Leu Ar g Pr o Ar g

   1 5 <210> 51 <211> 5 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> sor t ase r ecogni t i on mot i f <220>

   <221> mi sc_f eat ur e <222> (3)..(3) <223> Xaa can be any nat ur al l y occur r i ng ami no aci d <400> 51

   Leu Pr o Xaa Thr Gl y

   1 5 <210> 52 <211> 11

   Page 119

   2018203206 08 May 2018

   97047_1 <212> PRT <213> Artificial Sequence <220>

   <223> pol ypept i de <400> 52

   Pro Lys Asn Ser Ser Met I l e Ser Asn Thr Pro 1 5 10 <210> 53 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pol ypept i de <400> 53

   Hi s Gl n Ser Leu Gl y Thr Gl n

   1 5 <210> 54 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pol ypept i de

   Page 120

   97047_1

   2018203206 08 May 2018

   <400> Hi s Gl 1 54 n As n Leu Ser As p Gl y 5 Lys <210> 55 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> pol ypept i de <400> 55 Hi s Gl n As n I l e Ser As p Gl y Lys 1 5 <210> 56 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> pol ypept i de <400> 56 Val I l e Ser Ser Hi s Leu Gl y Gl n 1 5

   Page 121

   97047_1

   2018203206 08 May 2018

   <210> 57 <211> 4 <212> PRT <213> Art i f i ci al Sequence <220> <223> l i nker

   <220> <221> REPEAT <222> (1). . (4) <223> Repeat f r om 1 t o 100 t i mes <400> 57

   Gl y Gl y Gl y Ser <210> 58 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> l i nker <220>

   <221> REPEAT <222> (1)..(3) <223> May be r epeat ed 1 t o 100 t i mes

   Page 122

   97047_1 <220>

   2018203206 08 May 2018

   <221> REPEAT <222> (4). . (8) <223> May be r epeat ed 1 t o 100 t i mes <400> 58 Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser 1 5

   <210> 59 <211> 7 <212> PRT <213> Art i f i ci al Sequence

   <220> <223> l i nker <400> 59 Ser Gl y Gl y Ser Gl y Gl y Ser 1 5

   <210> 60 <211> 15 <212> PRT <213> Art i f i ci al Sequence

   <220> <223> l i nker <400> 60

   Page 123

   2018203206 08 May 2018

   97047_1

   Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Gl y 1 5 10 15 <210> 61 <211> 16 <212> PRT <213> Artificial Sequence <220>

   <223> l i nker <400> 61

   Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser 1 5 10 15 <210> 62 <211> 18 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> l i nker <400> 62

   Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Ser Gl y Gl y Ser Gl y 1 5 10 15

   Gl y Ser

   Page 124

   97047_1

   2018203206 08 May 2018

   <210> 63 <211> 15 <212> PRT <213> Art i f i ci al Sequence

   <220> <223> l i nker <400> 63 Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser 1 5 10 15

   <210> 64 <211> 5 <212> PRT <213> Art i f i ci al Sequence

   <220> <223> l i nker

   <220> <221> REPEAT <222> (1). . (5) <223> May be r epeat ed 1- 20 t i mes <400> 64 Gl y Gl y Gl y Gl y Ser

   Page 125

   97047_1

   2018203206 08 May 2018

   1 5 <210> 65 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <220> <221> MI SC_FEATURE <222> (4). . (5) <223> Cl eavage si t e <400> 65 Lys Leu Thr Ar g Al a Gl u Thr 1 5 <210> 66 <211> 7 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <220> <221> MI SC_FEATURE

   Page 126

   97047 1

   2018203206 08 May 2018

   <222> (4). . (5) <223> Cl eavage si t e <400> 66

   Asp Phe Thr Ar g Val Val Gl y 1 5

   <210> 67 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e

   <220>

   <221> MI SC FEATURE

   <222> (4). . (5) <223> Cl eavage <400> 67 Thr Met Thr Ar g 1

   Si t e

   I l e Val Gl y Gl y 5 <210>

   <211>

   <212>

   <213>

   PRT

   Art i f i ci al Sequence

   Page 127

   2018203206 08 May 2018

   97047_1 <220>

   <223> cl eavage si t e <220>

   <221> MI SC_FEATURE <222> ( 4) . . ( 5) <223> Cl eavage si t e <400> 68

   Ser Pr o Phe Ar g Ser Thr Gl y Gl y

   1 5 <210> 69 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <220>

   <221> MI SC_FEATURE <222> ( 4) . . ( 5) <223> Cl eavage si t e <400> 69

   Leu Gl n Val Ar g I l e Val Gl y Gl y

   1 5

   Page 128

   97047_1

   2018203206 08 May 2018 <210> 70 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <220>

   <221> MI SC_FEATURE <222> (4)..(5) <223> Cl eavage si t e <400> 70

   Pr o Leu Gl y Ar g I l e Val Gl y Gl y 1 5 <210> 71 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <220>

   <221> MI SC_FEATURE <222> ( 4) . . ( 5) <223> Cl eavage si t e

   Page 129

   2018203206 08 May 2018

   <400> 71 I l e Gl u Gl y Ar g Thr Val Gl y Gl y 1 5

   <210> 72 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e

   97047 1

   <220> <221> MI SC_FEATURE <222> (4). . (5) <223> cl eavage si t e <400> 72 Leu Thr Pr o Ar g Ser Leu Leu Val 1 5

   <210> 73 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e

   Page 130

   97047_1

   2018203206 08 May 2018 <220>

   <221> MI SC_FEATURE <222> (4)..(5) <223> cl eavage si t e <400> 73

   Leu Gl y Pr o Val Ser Gl y Val Pr o 1 5 <210> 74 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <220>

   <221> MI SC_FEATURE <222> ( 4) . . ( 5) <223> Cl eavage si t e <400> 74

   Val Al a Gl y Asp Ser Leu Gl u Gl u 1 5 <210> 75

   Page 131

   97047 1

   2018203206 08 May 2018

   <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e

   <220> <221> MI SC_FEATURE <222> (4). . (5) <223> Cl eavage si t e <400> 75 Gl y Pr o Al a Gl y Leu Gl y Gl y Al a 1 5

   <210> 76 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e

   <220> <221> MI SC_FEATURE <222> (4). . (5) <223> Cl eavage si t e <400> 76

   Page 132

   97047_1

   2018203206 08 May 2018

   Gl y Pr o Al a Gl y Leu Ar g Gl y Al a 1 5 <210> 77 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e <220>

   <221> MI SC_FEATURE <222> (4)..(5) <223> Cl eavage si t e <400> 77

   Al a Pr o Leu Gl y Leu Ar g Leu Ar g 1 5 <210> 78 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> cl eavage si t e

   Page 133

   97047_1

   2018203206 08 May 2018

   <220> <221> <222> <223> MI SC_FEATURE (4). . (5) Cl eavage si t e <400> 78 Pr o Al a Leu Pr o Leu Val Al a Gl n 1 5

   <210> 79 <211> 7 <212> PRT <213> Ar t i f i c i al Sequenc e <220> <223> cl eavage si t e

   <220> <221> <222> <223> MI SC_FEATURE (6). . (7) Cl eavage si t e <400> 79 Gl u As n ι Leu Tyr Phe Gl n Gl y 1 5

   <210> 80 <211> 8 <212> PRT

   Page 134

   97047 1

   2018203206 08 May 2018

   <213> Art i f i ci al Sequence <220> <223> cl eavage site <220> <221> MI SC_FEATURE <222> (4). . (5) <223> Cl eavage si t e <400> 80 Asp Asp Asp Lys I l e Val Gl y Gl y 1 5 <210> 81 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <220> <221> MI SC_FEATURE <222> (6). . (7) <223> Cl eavage si t e <400> 81 Leu Gl u Val Leu Phe Gl n Gl y Pr o

   Page 135

   97047_1

   2018203206 08 May 2018

   1 5 <210> 82 <211> 8 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <220> <221> MI SC_FEATURE <222> (4). . (5) <223> Cl eavage si t e <400> 82 Leu Pr o Lys Thr Gl y Ser Gl u Ser 1 5 <210> 83 <211> 9 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 83 Thr Gl n Ser Phe As n As p Phe Thr Ar g

   Page 136

   97047 1

   2018203206 08 May 2018

   1 5 <210> 84 <211> 10 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 84 Ser Val Ser Gl n Thr Ser Lys Leu Thr Ar g 1 5 10 <210> 85 <211> 10 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 85 Asp Phe Leu Al a Gl u Gl y Gl y Gl y Val Ar g 1 5 10 <210> 86 <211> 7 <212> PRT

   Page 137

   97047 1

   2018203206 08 May 2018

   <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 86 Thr Thr Ly s I l e Ly s Pr o Ar g 1 5 <210> 87 <211> 5 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 87 Leu Val Pro Arg Gl y 1 5 <210> 88 <211> 10 <212> PRT <213> Art i f i ci al Sequence <220> <223> cl eavage si t e <400> 88

   Page 138

   2018203206 08 May 2018

   97047_1

   Al a Leu Ar g Pr o Ar g Val Val Gl y Gl y Al a 1 5 10 <210> 89 <211> 1896 <212> PRT <213> Artificial Sequence <220>

   <223> FVI I I 198 <400> 89

   Met 1 Gl n I l e Gl u Leu Ser 5 Thr Cy s Phe Phe 10 Leu Cy s Leu Leu Ar g 15 Phe Cy s Phe Ser Al a Thr Ar g Ar g Ty r Ty r Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Ty r Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g 35 40 45 Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val 50 55 60 Ty r Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e 65 70 75 80

   Page 139

   97047 1

   2018203206 08 May 2018

   Al a Lys Pr o Ar g Pr o Pr o Tr p 85 Met Gl y Leu 90 Leu Gl y Pr o Thr I l e 95 Gl n Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser 100 105 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Ty r Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 Asp Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Ty r Val Tr p Gl n Val Leu 145 150 155 160 Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o Leu Cy s Leu Thr Ty r Ser 165 170 175 Tyr Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e 180 185 190 Gly Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205 Gl n Thr Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y

   Page 140

   2018203206 08 May 2018

   97047_1

   210 215 220

   Lys 225 Ser Tr p Hi s Ser Gl u 230 Thr Lys As n Ser Leu 235 Met Gl n As p Ar g As p 240 Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Ty r 245 250 255 Val Asn Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cy s Hi s Ar g Lys Ser Val 260 265 270 Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285 Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser 290 295 300 Leu Gl u I l e Ser Pr o Ile Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met 305 310 315 320 Asp Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s 325 330 335 Asp Gl y Met Gl u Al a Tyr Val Lys Val As p Ser Cy s Pr o Gl u Gl u Pr o 340 345 350

   Page 141

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   Gl n Leu Ar g 355 Met Ly s As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p 360 365 Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser 370 375 380 Pr o Ser Phe I l e Gl n Ile Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr 385 390 395 400 Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n 420 425 430 As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met 435 440 445 Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a I l e Gl n Hi s Gl u 450 455 460 Ser Gly I l e Leu Gl y Pr o Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu 465 470 475 480 Leu I l e I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n I l e Tyr Pr o

   Page 142

   2018203206 08 May 2018

   97047_1

   485 490 495

   Hi s Gl y I l e Thr 500 As p Val Ar g Pr o Leu 505 Tyr Ser Ar g Ar g Leu 510 Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe 515 520 525 Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p 530 535 540 Pr o Ar g Cys Leu Thr Ar g Tyr Tyr Ser Ser Phe Val As n Met Gl u Ar g 545 550 555 560 As p Leu Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cys Tyr Lys Gl u 565 570 575 Ser Val As p Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val 580 585 590 I l e Leu Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Tyr Leu Thr Gl u 595 600 605 As n I l e Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p 610 615 620

   Page 143

   97047_1

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   Pro G u 625 Phe Gl n Al a Ser 630 As n Ile Met Hi s Ser 635 Ile As n Gl y Ty r Val 640 Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p 645 650 655 Ty r I l e Leu Ser Ile Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe 660 665 670 Ser Gl y Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr 675 680 685 Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o 690 695 700 Gl y Leu Tr p I l e Leu Gl y Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y 705 710 715 720 Met Thr Al a Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p 725 730 735 Ty r Ty r Gl u As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys 740 745 750 As n As n Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Ser Ar g Hi s Pr o

   Page 144

   2018203206 08 May 2018

   97047_1

   755 760 765

   Ser Thr 770 Arg Gl n Lys Gl n Phe As n 775 Al a Thr Thr Ile 780 Pr o Gl u As n As p I l e Gl u Lys Thr As p Pr o Tr p Phe Al a Hi s Ar g Thr Pr o Met Pr o Lys 785 790 795 800 I l e Gl n As n Val Ser Ser Ser As p Leu Leu Met Leu Leu Ar g Gl n Ser 805 810 815 Pr o Thr Pr o Hi s Gl y Leu Ser Leu Ser As p Leu Gl n Gl u Al a Lys Tyr 820 825 830 Gl u Thr Phe Ser As p As p Pr o Ser Pr o Gl y Al a Ile As p Ser As n As n 835 840 845 Ser Leu Ser Gl u Met Thr Hi s Phe Ar g Pr o Gl n Leu Hi s Hi s Ser Gl y 850 855 860 Asp Met Val Phe Thr Pr o Gl u Ser Gl y Leu Gl n Leu Ar g Leu As n Gl u 865 870 875 880 Lys Leu Gl y Thr Thr Al a Al a Thr Gl u Leu Lys Lys Leu As p Phe Lys 885 890 895

   Page 145

   97047_1

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   Val Ser Ser Thr 900 Ser As n As n Leu Ile 905 Ser Thr Ile Pr o Ser 910 As p As n Leu Al a Al a Gl y Thr As p As n Thr Ser Ser Leu Gl y Pr o Pr o Ser Met 915 920 925 Pr o Val Hi s Tyr As p Ser Gl n Leu As p Thr Thr Leu Phe Gl y Lys Lys 930 935 940 Ser Ser Pr o Leu Thr Gl u Ser Gl y Gl y Pr o Leu Ser Leu Ser Gl u Gl u 945 950 955 960 As n As n As p Ser Lys Leu Leu Gl u Ser Gl y Leu Met As n Ser Gl n Gl u 965 970 975 Ser Ser Tr p Gl y Lys As n Val Ser Ser Gl u Ile Thr Ar g Thr Thr Leu 980 985 990 Gl n Ser As p Gl n Gl u Gl u Ile As p Ty r As p As p Thr I l e Ser Val Gl u 995 1000 1005

   Met Lys

   Lys Gl u Asp Phe Asp

   I l e Ty r As p Gl u As p

   Gl u As n Gl n

   1010

   1015

   1020

   Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Page 146

   2018203206 08 May 2018

   1025 1030 97047 _1 1035 Al a Val Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser Ser Pr o Hi s 1040 1045 1050 Val Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o Gl n Phe Lys 1055 1060 1065 Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe Thr Gl n Pr o 1070 1075 1080 Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu Leu Gl y Pr o 1085 1090 1095 Tyr I l e Ar g Al a Gl u Val Gl u As p As n Ile Met Val Thr Phe Ar g 1100 1105 1110 Asn Gl n Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser Ser Leu I l e Ser 1115 1120 1125 Tyr Gl u Gl u Asp Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g Lys As n Phe 1130 1135 1140 Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys Val Gl n Hi s 1145 1150 1155

   Page 147

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   Hi s Met 1160 Al a Pr o Thr Lys As p 1165 Gl u Phe As p Cys Lys 1170 Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u Lys As p Val Hi s Ser Gl y Leu 1175 1180 1185 I l e Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o Al a 1190 1195 1200 Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr 1205 1210 1215 I l e Phe As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u As n Met Gl u 1220 1225 1230 Ar g As n Cys Ar g Al a Pr o Cys As n Ile Gl n Met Gl u As p Pr o Thr 1235 1240 1245 Phe Lys Gl u As n Tyr Ar g Phe Hi s Al a Ile As n Gl y Tyr I l e Met 1250 1255 1260 Asp Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n Ar g I l e Ar g 1265 1270 1275 Tr p Tyr Leu Leu Ser Met Gl y Ser As n Gl u As n Ile Hi s Ser I l e

   Page 148

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   1280 1285 97047 _1 1290 Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Ty r 1295 1300 1305 Lys Met Al a Leu Ty r As n Leu Ty r Pr o Gl y Val Phe Gl u Thr Val 1310 1315 1320 Gl u Met Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cy s Leu 1325 1330 1335 I l e Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val 1340 1345 1350 Ty r Ser As n Lys Cy s Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s 1355 1360 1365 I l e Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Ty r Gl y Gl n Tr p 1370 1375 1380 Al a Pr o Lys Leu Al a Ar g Leu Hi s Ty r Ser Gl y Ser I l e As n Al a 1385 1390 1395 Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu 1400 1405 1410

   Page 149

   97047_1

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   Al a Pr o 1415 Met Ile Ile Hi s Gl y 1420 Ile Lys Thr Gl n Gl y 1425 Al a Ar g Gl n Lys Phe Ser Ser Leu Tyr Ile Ser Gl n Phe Ile Ile Met Tyr Ser 1430 1435 1440 Leu As p Gly Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y 1445 1450 1455 Thr Leu Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y I l e Lys 1460 1465 1470 Hi s As n I l e Phe Asn Pr o Pr o Ile Ile Al a Ar g Tyr I l e Ar g Leu 1475 1480 1485 Hi s Pr o Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu 1490 1495 1500 Met Gl y Cys Asp Leu As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u 1505 1510 1515 Ser Lys Al a Ile Ser As p Al a Gl n Ile Thr Al a Ser Ser Tyr Phe 1520 1525 1530 Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s

   Page 150

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   1535 1540 97047 _1 1545 Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o 1550 1555 1560 Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr 1565 1570 1575 Gl y Val Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Tyr 1580 1585 1590 Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p 1595 1600 1605 Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n 1610 1615 1620 Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu 1625 1630 1635 Leu Thr Ar g Tyr Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n 1640 1645 1650 I l e Al a Leu Ar g Met Gl u Val Leu Gl y Cys Gl u Al a Gl n As p Leu 1655 1660 1665

   Page 151

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   97047 1

   Tyr As p 1670 Lys Thr Hi s Thr Cys 1675 Pr o Pr o Cys Pr o Al a 1680 Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p 1685 1690 1695 Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val Val 1700 1705 1710 Asp Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Tyr Val 1715 1720 1725 Asp Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u 1730 1735 1740 Gl n Tyr As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu 1745 1750 1755 Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser 1760 1765 1770 As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a 1775 1780 1785 Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser

   Page 152

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   1790 Gl u 1795 Gl n Val Ser Leu 1800 Leu Val Ar g As p 1805 Leu Thr Lys As n 1810 Thr 1815 Cy s Lys Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n 1820 1825 1830 Gl y Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p 1835 1840 1845 Ser As p Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys 1850 1855 1860 Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s 1865 1870 1875 Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser 1880 1885 1890

   Pr o Gl y Lys 1895

   <210> 90 <211> 99 <212> DNA

   Page 153

   2018203206 08 May 2018

   97047_1 <213> Art i f i ci al Sequence <220>

   <223> ESC48- Fwd - VWF- D D3 wi t h VI I I si gnal and Bsi W1 site <400> 90 t cgcgacgt a cggccgccac cat gcaaat a gagct ct cca cctgcttctt tctgtgcctt 60 ttgcgattct gctttagcct at cct gt cgg ccccccat g 99 <210> 91 <211> 74 <212> DNA <213> Art i f i ci al Sequence <220>

   <223> ESC51- Rev- VWF D' D3 ( 1- 477 ami no aci d) wi t h 6Hi s and Not 1 si t e <400> 91 t gacct cgag cggccgct ca gt ggt gat gg t gat gat gcg gct cct ggca ggct t cacag 60 gt gaggt t ga caac 74

   <210> 92 <211> 32 <212> DNA <213> Ar t i f i c i al Sequence <220> <223> ESC 89-fwd wi t h Nhe1si t e <400> 92

   Page 154

   2018203206 08 May 2018

   97047_1 ct cact at ag ggagacccaa gct ggct agc cg 32 <210> 93 <211> 43 <212> DNA <213> Artificial Sequence <220>

   <223> ESC 91- r ev wi t h Sal 1 <400> 93 ct ggat cccg ggagt cgact cgt cagt ggt gat ggt gat g at g 43 <210> 94 <211> 40 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> l i nker <400> 94

   Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

   1 5 10 15

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y

   Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser

   Page 155

   97047_1

   35 40

   2018203206 08 May 2018

   <210> 95 <211> 92 <212> DNA <213> Art i f i ci al Sequence <220> <223> LW22- FWD VWF- D D3 wi t h FVIII si gnal sequence and Bsi W site <400> 95 gcgccggccg t acgat gcaa at agagct ct ccacct gct t ct t t ct gt gc ct t t t gcgat tctgctttag cct at cct gt cggcccccca tg <210> 96 <211> 47 <212> DNA <213> Ar t i f i c i al Sequenc e <220> <223> LW23-Rev- Fc with stop codon and Not 1 site <400> 96 t cat caat gt at ct t at cat gt ct gaat t c gcggccgct c at t t acc <210> 97 <211> 41 <212> DNA <213> Ar t i f i c i al Sequenc e

   Page 156

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   97047_1 <220>

   <223> LW24- Fwd- VWF D1 D2D D3 cl oni ng ol i go wi t h Bsi W1 site <400> 97 gcgccggccg tacgatgatt cct gccagat ttgccggggt g 41 <210> 98 <211> 41 <212> DNA <213> Art i f i ci al Sequence <220>

   <223> LW27- Rev- VWF D' D3 ol i go wi t h EcoRV <400> 98 ccaccgccag at atcggctc ctggcaggct tcacaggtga g 41 <210> 99 <211> 1240 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> VWF- D1D2D' D3 pr ot ei n sequence 1 <400> 99

   Met I l e Pr o Al a Ar g Phe Al a Gl y Val Leu Leu Al a Leu Al a Leu I l e

   1 5 10 15

   Leu Pr o Gl y Thr Leu Cy s Al a Gl u Gl y Thr Ar g Gl y Ar g Ser Ser Thr

   Page 157

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   20 25 97047_1 30 Al a Ar g Cy s Ser Leu Phe Gl y Ser As p Phe Val As n Thr Phe As p Gl y 35 40 45 Ser Met Ty r Ser Phe Al a Gl y Ty r Cy s Ser Ty r Leu Leu Al a Gl y Gl y 50 55 60 Cy s Gl n Lys Ar g Ser Phe Ser Ile Ile Gl y As p Phe Gl n As n Gl y Lys 65 70 75 80 Ar g Val Ser Leu Ser Val Ty r Leu Gl y Gl u Phe Phe As p I l e Hi s Leu 85 90 95 Phe Val As n Gl y Thr Val Thr Gl n Gl y As p Gl n Ar g Val Ser Met Pr o 100 105 110 Ty r Al a Ser Lys Gl y Leu Ty r Leu Gl u Thr Gl u Al a Gl y Ty r Ty r Lys 115 120 125 Leu Ser Gl y Gl u Al a Ty r Gl y Phe Val Al a Ar g Ile As p Gl y Ser Gl y 130 135 140 As n Phe Gl n Val Leu Leu Ser As p Ar g Ty r Phe As n Lys Thr Cy s Gl y 145 150 155 160

   Page 158

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   97047 1

   Leu Cys Gl y As n Phe As n 165 Ile Phe Al a Gl u 170 As p As p Phe Met Thr 175 Gl n Gl u Gl y Thr Leu Thr Ser As p Pr o Tyr As p Phe Al a As n Ser Tr p Al a 180 185 190 Leu Ser Ser Gl y Gl u Gl n Tr p Cys Gl u Ar g Al a Ser Pr o Pr o Ser Ser 195 200 205 Ser Cys As n I l e Ser Ser Gl y Gl u Met Gl n Lys Gl y Leu Tr p Gl u Gl n 210 215 220 Cys Gl n Leu Leu Lys Ser Thr Ser Val Phe Al a Ar g Cys Hi s Pr o Leu 225 230 235 240 Val As p Pr o Gl u Pr o Phe Val Al a Leu Cys Gl u Lys Thr Leu Cys Gl u 245 250 255 Cys Al a Gl y Gl y Leu Gl u Cys Al a Cys Pr o Al a Leu Leu Gl u Tyr Al a 260 265 270 Ar g Thr Cys Al a Gl n Gl u Gl y Met Val Leu Tyr Gl y Tr p Thr As p Hi s 275 280 285 Ser Al a Cys Ser Pr o Val Cys Pr o Al a Gl y Met Gl u Tyr Ar g Gl n Cys

   Page 159

   2018203206 08 May 2018

   97047_1

   290 295 300

   Val Ser Pr o Cys Al a Ar g Thr Cy s Gl n Ser Leu Hi s I l e As n Gl u Met 305 310 315 320 Cys Gl n Gl u Ar g Cys Val As p Gl ^y Cys Ser Cys Pr o Gl u Gl y Gl n Leu 325 330 335 Leu As p Gl u Gl y Leu Cys Val Gl u Ser Thr Gl u Cys Pr o Cys Val Hi s 340 345 350 Ser Gl y Lys Ar g Tyr Pr o Pr o Gl ^y Thr Ser Leu Ser Ar g As p Cys As n 355 36 0 365 Thr Cys I l e Cys Ar g As n Ser Gl n Tr p Ile Cys Ser As n Gl u Gl u Cys 370 375 380 Pr o Gl y Gl u Cys Leu Val Thr Gl ^y Gl n Ser Hi s Phe Lys Ser Phe As p 385 390 395 400 As n Ar g Tyr Phe Thr Phe Ser Gl ^y Ile Cys Gl n Tyr Leu Leu Al a Ar g 405 410 415 As p Cys Gl n As p Hi s Ser Phe Se r Ile Val Ile Gl u Thr Val Gl n Cys 420 425 430

   Page 160

   97047_1

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   Al a As p As p 435 Ar g As p Al a Val Cy s 440 Thr Ar g Ser Val Thr 445 Val Ar g Leu Pr o Gl y Leu Hi s As n Ser Leu Val Lys Leu Lys Hi s Gl y Al a Gl y Val 450 455 460 Al a Met As p Gl y Gl n As p Ile Gl n Leu Pr o Leu Leu Lys Gl y As p Leu 465 470 475 480 Ar g I l e Gl n Hi s Thr Val Thr Al a Ser Val Ar g Leu Ser Ty r Gl y Gl u 485 490 495 As p Leu Gl n Met As p Tr p As p Gl y Ar g Gl y Ar g Leu Leu Val Lys Leu 500 505 510 Ser Pr o Val Ty r Al a Gl y Lys Thr Cy s Gl y Leu Cy s Gl y As n Ty r As n 515 520 525 Gl y As n Gl n Gl y As p As p Phe Leu Thr Pr o Ser Gl y Leu Al a Gl u Pr o 530 535 540 Ar g Val Gl u As p Phe Gl y As n Al a Tr p Lys Leu Hi s Gl y As p Cy s Gl n 545 550 555 560 As p Leu Gl n Lys Gl n Hi s Ser As p Pr o Cy s Al a Leu As n Pr o Ar g Met

   Page 161

   2018203206 08 May 2018

   97047_1

   565 570 575

   Thr Ar g Phe Ser 580 Gl u Gl u Al a Cys Al a 585 Val Leu Thr Ser Pr o 590 Thr Phe Gl u Al a Cys Hi s Ar g Al a Val Ser Pr o Leu Pr o Tyr Leu Ar g As n Cys 595 600 605 Ar g Tyr As p Val Cys Ser Cys Ser As p Gl y Ar g Gl u Cys Leu Cys Gl y 610 615 620 Al a Leu Al a Ser Tyr Al a Al a Al a Cys Al a Gl y Ar g Gl y Val Ar g Val 625 630 635 640 Al a Tr p Ar g Gl u Pr o Gl y Ar g Cys Gl u Leu As n Cys Pr o Lys Gl y Gl n 645 650 655 Val Tyr Leu Gl n Cys Gl y Thr Pr o Cys As n Leu Thr Cys Ar g Ser Leu 660 665 670 Ser Tyr Pr o As p Gl u Gl u Cys As n Gl u Al a Cys Leu Gl u Gl y Cys Phe 675 680 685 Cys Pr o Pr o Gl y Leu Tyr Met As p Gl u Ar g Gl y As p Cys Val Pr o Lys 690 695 700

   Page 162

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   Al a Gl n 705 Cy s Pr o Cy s Ty r 710 Ty r As p Gl y Gl u Ile 715 Phe Gl n Pr o Gl u As p 720 I l e Phe Ser As p Hi s Hi s Thr Met Cy s Ty r Cy s Gl u As p Gl y Phe Met 725 730 735 Hi s Cy s Thr Met Ser Gl y Val Pr o Gl y Ser Leu Leu Pr o As p Al a Val 740 745 750 Leu Ser Ser Pr o Leu Ser Hi s Ar g Ser Lys Ar g Ser Leu Ser Cy s Ar g 755 760 765 Pr o Pr o Met Val Lys Leu Val Cy s Pr o Al a As p As n Leu Ar g Al a Gl u 770 775 780 Gl y Leu Gl u Cy s Thr Lys Thr Cy s Gl n As n Ty r As p Leu Gl u Cy s Met 785 790 795 800 Ser Met Gl y Cy s Val Ser Gl y Cy s Leu Cy s Pr o Pr o Gl y Met Val Ar g 805 810 815 Hi s Gl u As n Ar g Cy s Val Al a Leu Gl u Ar g Cy s Pr o Cy s Phe Hi s Gl n 820 825 830 Gl y Lys Gl u Ty r Al a Pr o Gl y Gl u Thr Val Lys Ile Gl y Cy s As n Thr

   Page 163

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   97047_1

   835 840 845

   Cys Val Cy s Ar g As p Ar g Lys 855 Tr p As n Cy s Thr As p 860 Hi s Val Cy s As p 850 Al a Thr Cy s Ser Thr Ile Gl y Met Al a Hi s Ty r Leu Thr Phe As p Gl y 865 870 875 880 Leu Lys Ty r Leu Phe Pr o Gl y Gl u Cy s Gl n Ty r Val Leu Val Gl n As p 885 890 895 Ty r Cy s Gl y Ser As n Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys 900 905 910 Gl y Cy s Ser Hi s Pr o Ser Val Lys Cy s Lys Lys Ar g Val Thr I l e Leu 915 920 925 Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y Gl u Val As n Val Lys 930 935 940 Ar g Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g 945 950 955 960 Ty r I l e I l e Leu Leu Leu Gl y Lys Al a Leu Ser Val Val Tr p As p Ar g 965 970 975

   Page 164

   2018203206 08 May 2018

   97047_1

   Hi s Leu Ser I l e Ser Val Val Leu Lys Gl n Thr Tyr Gl n Gl u Lys Val 980 985 990

   Cys Gl y Leu Cys Gl y Asn Phe Asp Gl y Ile Gl n Asn Asn Asp Leu Thr 995 1000 1005

   Ser Ser 1010 As n Leu Gl n Val Gl u 1015 Gl u As p Pr o Val As p 1020 Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cy s Al a As p Thr Ar g Lys Val Pr o 1025 1030 1035 Leu As p Ser Ser Pr o Al a Thr Cy s Hi s As n As n Ile Met Lys Gl n 1040 1045 1050 Thr Met Val As p Ser Ser Cy s Ar g Ile Leu Thr Ser As p Val Phe 1055 1060 1065 Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu As p Val 1070 1075 1080 Cy s I l e Ty r As p Thr Cy s Ser Cy s Gl u Ser Ile Gl y As p Cy s Al a 1085 1090 1095 Cy s Phe Cy s As p Thr Ile Al a Al a Ty r Al a Hi s Val Cy s Al a Gl n

   Page 165

   2018203206 08 May 2018

   1100 1105 97047 _1 1110 Hi s Gl y Lys Val Val Thr Tr p Ar g Thr Al a Thr Leu Cy s Pr o Gl n 1115 1120 1125 Ser Cys Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Ty r Gl u Cy s Gl u 1130 1135 1140 Tr p Ar g Ty r As n Ser Cy s Al a Pr o Al a Cy s Gl n Val Thr Cy s Gl n 1145 1150 1155 Hi s Pr o Gl u Pr o Leu Al a Cy s Pr o Val Gl n Cy s Val Gl u Gl y Cy s 1160 1165 1170 Hi s Al a Hi s Cy s Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu Leu Gl n 1175 1180 1185 Thr Cy s Val As p Pr o Gl u As p Cy s Pr o Val Cy s Gl u Val Al a Gl y 1190 1195 1200 Ar g Ar g Phe Al a Ser Gl y Lys Lys Val Thr Leu As n Pr o Ser As p 1205 1210 1215 Pr o Gl u Hi s Cy s Gl n Ile Cy s Hi s Cy s As p Val Val As n Leu Thr 1220 1225 1230

   Page 166

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   2018203206 08 May 2018

   Cys Gl u Al a Cys Gl n Gl u Pro 1235 1240 <210> 100 <211> 477 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> VWF- D' D3 pr ot ei n sequence 2 <400> 100

   Ser 1 Leu Ser Cys Ar g 5 Pr o Pr o Met Val Lys 10 Leu Val Cys Pr o Al a As p 15 As n Leu Ar g Al a Gl u Gl y Leu Gl u Cys Thr Lys Thr Cys Gl n As n Tyr 20 25 30 As p Leu Gl u Cys Met Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o 35 40 45 Pr o Gl y Met Val Ar g Hi s Gl u As n Ar g Cys Val Al a Leu Gl u Ar g Cys 50 55 60 Pr o Cys Phe Hi s Gl n Gl y Lys Gl u Tyr Al a Pr o Gl y Gl u Thr Val Lys 65 70 75 80

   Page 167

   97047_1

   2018203206 08 May 2018

   I l e Gl y Cy s As n Thr 85 Cy s Val Cy s Ar g As p Ar g 90 Lys Tr p As n Cy s 95 Thr Asp Hi s Val Cy s As p Al a Thr Cy s Ser Thr Ile Gl y Met Al a Hi s Ty r 100 105 110 Leu Thr Phe As p Gl y Leu Lys Ty r Leu Phe Pr o Gl y Gl u Cy s Gl n Ty r 115 120 125 Val Leu Val Gl n As p Ty r Cy s Gl y Ser As n Pr o Gl y Thr Phe Ar g I l e 130 135 140 Leu Val Gl y As n Lys Gl y Cy s Ser Hi s Pr o Ser Val Lys Cy s Lys Lys 145 150 155 160 Ar g Val Thr I l e Leu Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y 165 170 175 Gl u Val As n Val Lys Ar g Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val 180 185 190 Val Gl u Ser Gl y Ar g Ty r Ile Ile Leu Leu Leu Gl y Lys Al a Leu Ser 195 200 205 Val Val Tr p As p Ar g Hi s Leu Ser Ile Ser Val Val Leu Lys Gl n Thr

   Page 168

   2018203206 08 May 2018

   97047_1

   210 215 220

   Ty r 225 Gl n Gl u Lys Val Cy s 230 Gl y Leu Cy s Gl y As n 235 Phe As p Gl y I l e Gl n 240 As n As n As p Leu Thr Ser Ser As n Leu Gl n Val Gl u Gl u As p Pr o Val 245 250 255 As p Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cy s Al a As p Thr Ar g 260 265 270 Lys Val Pr o Leu As p Ser Ser Pr o Al a Thr Cy s Hi s As n As n I l e Met 275 280 285 Lys Gl n Thr Met Val As p Ser Ser Cy s Ar g Ile Leu Thr Ser As p Val 290 295 300 Phe Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu As p Val 305 310 315 320 Cy s I l e Ty r As p Thr Cy s Ser Cy s Gl u Ser Ile Gl y As p Cy s Al a Cy s 325 330 335 Phe Cy s As p Thr Ile Al a Al a Ty r Al a Hi s Val Cy s Al a Gl n Hi s Gl y 340 345 350

   Page 169

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   Lys Val Val 355 Thr Tr p Ar g Thr Al a 360 Thr Leu Cys Pr o Gl n 365 Ser Cys Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Tyr Gl u Cys Gl u Tr p Ar g Tyr As n 370 375 380 Ser Cys Al a Pr o Al a Cys Gl n Val Thr Cys Gl n Hi s Pr o Gl u Pr o Leu 385 390 395 400 Al a Cys Pr o Val Gl n Cys Val Gl u Gl y Cys Hi s Al a Hi s Cys Pr o Pr o 405 410 415 Gl y Lys I l e Leu As p Gl u Leu Leu Gl n Thr Cys Val As p Pr o Gl u As p 420 425 430 Cys Pr o Val Cys Gl u Val Al a Gl y Ar g Ar g Phe Al a Ser Gl y Lys Lys 435 440 445 Val Thr Leu As n Pr o Ser As p Pr o Gl u Hi s Cys Gl n I l e Cys Hi s Cys 450 455 460

   Asp Val Val Asn Leu Thr Cys Gl u Al a Cys Gl n Gl u Pro

   465

   470

   475 <210> 101

   Page 170

   2018203206 08 May 2018

   97047_1 <211> 2754 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN-FVI I I - 161 <400> 101

   Met 1 Gl n I l e Gl u Leu Ser 5 Thr Cys Phe Phe 10 Leu Cys Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g 35 40 45 Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e 65 70 75 80 Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr I l e Gl n 85 90 95 Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser

   Page 171

   2018203206 08 May 2018

   100 105 97047_1 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu 145 150 155 160 Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e 180 185 190 Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205 Gl n Thr Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y 210 215 220 Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p 225 230 235 240

   Page 172

   97047 1

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   Al a Al a Ser Al a Ar g Al a 245 Tr p Pr o Lys Met 250 Hi s Thr Val As n Gl y 255 Tyr Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val 260 265 270 Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285 Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser 290 295 300 Leu Gl u I l e Ser Pr o Ile Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met 305 310 315 320 As p Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s 325 330 335 As p Gl y Met Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o 340 345 350 Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Tyr As p As p As p 355 360 365 Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser

   Page 173

   2018203206 08 May 2018

   97047_1

   370 375 380

   Pr o 385 Ser Phe I l e Gl n Ile 390 Ar g Ser Val Al a Lys 395 Lys Hi s Pr o Lys Thr 400 Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n 420 425 430 As n Gly Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met 435 440 445 Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a I l e Gl n Hi s Gl u 450 455 460 Ser Gl y I l e Leu Gl y Pr o Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu 465 470 475 480 Leu I l e I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n I l e Tyr Pr o 485 490 495 Hi s Gl y I l e Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys 500 505 510

   Page 174

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   Gl y Val Lys 515 Hi s Leu Lys Asp Phe 520 Pr o Ile Leu Pr o Gl y 525 Gl u I l e Phe Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p 530 535 540 Pr o Ar g Cys Leu Thr Ar g Ty r Ty r Ser Ser Phe Val As n Met Gl u Ar g 545 550 555 560 Asp Leu Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cy s Ty r Lys Gl u 565 570 575 Ser Val As p Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val 580 585 590 I l e Leu Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u 595 600 605 As n I l e Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p 610 615 620 Pr o Gl u Phe Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val 625 630 635 640 Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p

   Page 175

   2018203206 08 May 2018

   97047_1

   645 650 655

   Ty r I l e Leu Ser 660 Ile Gl y Al a Gl n Thr 665 As p Phe Leu Ser Val 670 Phe Phe Ser Gl y Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr 675 680 685 Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o 690 695 700 Gl y Leu Tr p I l e Leu Gl y Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y 705 710 715 720 Met Thr Al a Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p 725 730 735 Ty r Ty r Gl u As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys 740 745 750 As n As n Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Pr o Pr o Val Leu 755 760 765 Lys Ar g Hi s Gl n Ar g Gl u Ile Thr Ar g Thr Thr Leu Gl n Ser As p Gl n 770 775 780

   Page 176

   97047_1

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   Gl u 785 Gl u I l e Asp Tyr As p 790 As p Thr Ile Ser Val 795 Gl u Met Lys Lys Gl u 800 As p Phe As p I l e Tyr As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe 805 810 815 Gl n Lys Lys Thr Ar g Hi s Tyr Phe Ile Al a Al a Val Gl u Ar g Leu Tr p 820 825 830 As p Tyr Gl y Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n 835 840 845 Ser Gl y Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr 850 855 860 As p Gl y Ser Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s 865 870 875 880 Leu Gl y Leu Leu Gl y Pr o Tyr Ile Ar g Al a Gl u Val Gl u As p As n I l e 885 890 895 Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser 900 905 910 Ser Leu I l e Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g

   Page 177

   2018203206 08 May 2018

   915 920 97047_1 925 Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys Val 930 935 940 Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys Al a Tr p 945 950 955 960 Al a Tyr Phe Ser As p Val As p Leu Gl u Lys As p Val Hi s Ser Gl y Leu 965 970 975 I l e Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s 980 985 990 Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe

   995 1000 1005

   As p Gl u

   Thr Lys Ser Trp Tyr

   Phe Thr

   Gl u Asn Met

   Gl u Ar g As n

   1010

   1015

   1020

   Cys Arg

   Al a Pro Cys Asn Ile

   Gl n Met Gl u Asp Pr o

   Thr Phe Lys

   1025

   1030

   1035

   Gl u As n

   Tyr Arg Phe Hi s Al a

   I l e As n Gl y Ty r I l e

   Met Asp Thr

   1040

   1045

   1050

   Page 178

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   Leu Pr o 1055 Gl y Leu Val Met Al a 1060 Gl n As p Gl n Ar g Ile 1065 Ar g Tr p Ty r Leu Leu Ser Met Gl y Ser As n Gl u As n Ile Hi s Ser I l e Hi s Phe 1070 1075 1080 Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Ty r Lys Met 1085 1090 1095 Al a Leu Ty r As n Leu Ty r Pr o Gl y Val Phe Gl u Thr Val Gl u Met 1100 1105 1110 Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cy s Leu I l e Gl y 1115 1120 1125 Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Ty r Ser 1130 1135 1140 As n Lys Cy s Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g 1145 1150 1155 As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Ty r Gl y Gl n Tr p Al a Pr o 1160 1165 1170 Lys Leu Al a Arg Leu Hi s Ty r Ser Gl y Ser Ile As n Al a Tr p Ser

   Page 179

   2018203206 08 May 2018

   1175 1180 97047 _1 1185 Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o 1190 1195 1200 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe 1205 1210 1215 Ser Ser Leu Tyr Ile Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p 1220 1225 1230 Gl y Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu 1235 1240 1245 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n 1250 1255 1260 I l e Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr Ile Ar g Leu Hi s Pr o 1265 1270 1275 Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y 1280 1285 1290 Cys As p Leu As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys 1295 1300 1305

   Page 180

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   Al a I l e 1310 Ser As p Al a Gl n Ile 1315 Thr Al a Ser Ser Ty r 1320 Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n 1325 1330 1335 Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u 1340 1345 1350 Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1355 1360 1365 Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Ty r Val Lys 1370 1375 1380 Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu 1385 1390 1395 Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p 1400 1405 1410 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1415 1420 1425 Ar g Ty r Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n I l e Al a

   Page 181

   2018203206 08 May 2018

   1430 1435 97047 _1 1440 Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p Leu Ty r As p 1445 1450 1455 Lys Thr Hi s Thr Cy s Pr o Pr o Cy s Pr o Al a Pr o Gl u Leu Leu Gl y 1460 1465 1470 Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1475 1480 1485 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cy s Val Val Val As p Val 1490 1495 1500 Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Ty r Val As p Gl y 1505 1510 1515 Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Ty r 1520 1525 1530 As n Ser Thr Ty r Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n 1535 1540 1545 As p Tr p Leu As n Gl y Lys Gl u Ty r Lys Cy s Lys Val Ser As n Lys 1550 1555 1560

   Page 182

   97047_1

   2018203206 08 May 2018

   Al a Leu 1565 Pr o Al a Pr o Ile Gl u 1570 Lys Thr Ile Ser Lys 1575 Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p 1580 1585 1590 Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y 1595 1600 1605 Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n 1610 1615 1620 Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p 1625 1630 1635 Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g 1640 1645 1650 Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a 1655 1660 1665 Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y 1670 1675 1680 Lys Ar g Ar g Ar g Ar g Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y

   Page 183

   2018203206 08 May 2018

   1685 1690 97047 _1 1695 Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y 1700 1705 1710 Ser Gl y Gl y Gl y Gl y Ser Ar g Lys Ar g Ar g Lys Ar g Ser Leu Ser 1715 1720 1725 Cy s Ar g Pr o Pr o Met Val Lys Leu Val Cy s Pr o Al a As p As n Leu 1730 1735 1740 Ar g Al a Gl u Gl y Leu Gl u Cy s Thr Lys Thr Cy s Gl n As n Ty r As p 1745 1750 1755 Leu Gl u Cy s Met Ser Met Gl y Cy s Val Ser Gl y Cy s Leu Cy s Pr o 1760 1765 1770 Pr o Gl y Met Val Ar g Hi s Gl u As n Ar g Cy s Val Al a Leu Gl u Ar g 1775 1780 1785 Cy s Pr o Cy s Phe Hi s Gl n Gl y Lys Gl u Ty r Al a Pr o Gl y Gl u Thr 1790 1795 1800 Val Lys I l e Gl y Cy s As n Thr Cy s Val Cy s Ar g As p Ar g Lys Tr p 1805 1810 1815

   Page 184

   97047_1

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   As n Cy s 1820 Thr As p Hi s Val Cy s 1825 As p Al a Thr Cy s Ser 1830 Thr I l e Gl y Met Al a Hi s Ty r Leu Thr Phe As p Gl y Leu Lys Ty r Leu Phe Pr o 1835 1840 1845 Gl y Gl u Cy s Gl n Ty r Val Leu Val Gl n As p Ty r Cy s Gl y Ser As n 1850 1855 1860 Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys Gl y Cy s Ser Hi s 1865 1870 1875 Pr o Ser Val Lys Cy s Lys Lys Ar g Val Thr Ile Leu Val Gl u Gl y 1880 1885 1890 Gl y Gl u I l e Gl u Leu Phe As p Gl y Gl u Val As n Val Lys Ar g Pr o 1895 1900 1905 Met Lys As p Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g Ty r 1910 1915 1920 I l e I l e Leu Leu Leu Gl y Lys Al a Leu Ser Val Val Tr p As p Ar g 1925 1930 1935 Hi s Leu Ser Ile Ser Val Val Leu Lys Gl n Thr Ty r Gl n Gl u Lys

   Page 185

   2018203206 08 May 2018

   1940 1945 97047 _1 1950 Val Cy s Gl y Leu Cy s Gl y As n Phe As p Gl y Ile Gl n As n As n As p 1955 1960 1965 Leu Thr Ser Ser As n Leu Gl n Val Gl u Gl u As p Pr o Val As p Phe 1970 1975 1980 Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cy s Al a As p Thr Ar g Lys 1985 1990 1995 Val Pr o Leu As p Ser Ser Pr o Al a Thr Cy s Hi s As n As n I l e Met 2000 2005 2010 Lys Gl n Thr Met Val As p Ser Ser Cy s Ar g Ile Leu Thr Ser As p 2015 2020 2025 Val Phe Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu 2030 2035 2040 As p Val Cy s Ile Ty r As p Thr Cy s Ser Cy s Gl u Ser I l e Gl y As p 2045 2050 2055 Cy s Al a Al a Phe Cy s As p Thr Ile Al a Al a Ty r Al a Hi s Val Cy s 2060 2065 2070

   Page 186

   97047_1

   2018203206 08 May 2018

   Al a Gl n 2075 Hi s Gl y Lys Val Val 2080 Thr Tr p Ar g Thr Al a 2085 Thr Leu Cy s Pr o Gl n Ser Cy s Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Ty r Gl u 2090 2095 2100 Al a Gl u Tr p Ar g Ty r As n Ser Cy s Al a Pr o Al a Cy s Gl n Val Thr 2105 2110 2115 Cy s Gl n Hi s Pr o Gl u Pr o Leu Al a Cy s Pr o Val Gl n Cy s Val Gl u 2120 2125 2130 Gl y Cy s Hi s Al a Hi s Cy s Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu 2135 2140 2145 Leu Gl n Thr Cy s Val As p Pr o Gl u As p Cy s Pr o Val Cy s Gl u Val 2150 2155 2160 Al a Gl y Ar g Ar g Phe Al a Ser Gl y Lys Lys Val Thr Leu As n Pr o 2165 2170 2175 Ser As p Pr o Gl u Hi s Cy s Gl n Ile Cy s Hi s Cy s As p Val Val As n 2180 2185 2190 Leu Thr Cy s Gl u Al a Cy s Gl n Gl u Pr o Ile Ser Gl y Thr Ser Gl u

   Page 187

   97047_1

   2205

   2018203206 08 May 2018

   2195 2200 Ser Al a Thr Pr o Gl u Ser Gl y 2210 2215 Gl y Ser Gl u Thr Pr o Gl y Thr 2225 2230

   Pr o Gl y Ser Gl u Pr o Al a Thr Ser 2220

   Ser Gl u Ser Al a Thr Pr o Gl u Ser

   2235

   Gl y Pr o

   Gl y Ser Gl u Pr o Al a

   Thr Ser Gl y Ser Gl u

   Thr Pr o Gl y

   2240

   2245

   2250

   Thr Ser

   Gl u Ser Al a Thr Pr o

   Gl u Ser Gl y Pr o Gl y

   Thr Ser Thr

   2255

   2260

   2265

   Gl u Pr o

   Ser Gl u Gl y Ser Al a

   Pr o Gl y Ser Pr o Al a

   Gl y Ser Pr o

   2270

   2275

   2280

   Thr Ser

   Thr Gl u Gl u Gl y Thr

   Ser

   Gl u Ser Al a Thr

   Pr o Gl u Ser

   2285

   2290

   2295

   Gl y Pr o

   Gl y Ser Gl u Pr o Al a

   Thr Ser Gl y Ser Gl u

   Thr Pr o Gl y

   2300

   2305

   2310

   Thr Ser

   Gl u Ser Al a Thr Pr o

   Gl u Ser Gl y Pr o Gl y

   Ser Pr o Al a

   2315

   2320

   2325

   Page 188

   97047_1

   2018203206 08 May 2018

   Gl y Ser 2330 Pr o Thr Ser Thr Gl u 2335 Gl u Gl y Ser Pr o Al a 2340 Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser 2345 2350 2355 Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 2360 2365 2370 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u 2375 2380 2385 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser 2390 2395 2400 Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u 2405 2410 2415 Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y 2420 2425 2430 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 2435 2440 2445 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o Al a Thr Ser

   Page 189

   2018203206 08 May 2018

   97047_1

   2450 2455 2460

   Gly Ser 2465 Gl u Thr Pr o Gl y Thr 2470 Ser Gl u Ser Al a Thr 2475 Pr o Gl u Ser Gly Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o As p 2480 2485 2490 Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y 2495 2500 2505 Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Leu Val Pr o Ar g 2510 2515 2520 Gl y Ser Gl y Gl y As p Lys Thr Hi s Thr Cys Pr o Pr o Cys Pr o Al a 2525 2530 2535 Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys 2540 2545 2550 Pr o Lys Asp Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cys 2555 2560 2565 Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n

   2570 2575 2580

   Page 190

   97047_1

   2018203206 08 May 2018

   Tr p Tyr 2585 Val As p Gl y Val Gl u 2590 Val Hi s As n Al a Lys 2595 Thr Lys Pr o Arg Gl u Gl u Gl n Tyr As n Ser Thr Tyr Ar g Val Val Ser Val Leu 2600 2605 2610 Thr Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys 2615 2620 2625 Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr I l e 2630 2635 2640 Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu 2645 2650 2655 Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr 2660 2665 2670 Cys Leu Val Lys Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p 2675 2680 2685 Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o 2690 2695 2700 Val Leu As p Ser As p Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr

   Page 191

   2018203206 08 May 2018

   2705 2710 97047 _1 2715 Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser 2720 2725 2730 Val Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu 2735 2740 2745 Ser Leu Ser Pr o Gl y Lys

   2750 <210> 102 <211> 2242 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN- FVI I I - 170 pr ot ei n sequence <400> 102

   Ser 1 Leu Ser Cys Ar g 5 Pr o Pr o Met Val Lys 10 Leu Val Cys Pr o Al a 15 As p As n Leu Ar g Al a Gl u Gl y Leu Gl u Cys Thr Lys Thr Cys Gl n As n Tyr 20 25 30 As p Leu Gl u Cys Met Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o

   Page 192

   2018203206 08 May 2018

   97047_1

   35 40 45

   Pr o Gl y 50 Met Val Ar g Hi s Gl u As n 55 Ar g Cy s Val Al a 60 Leu Gl u Ar g Cy s Pr o Cy s Phe Hi s Gl n Gl y Lys Gl u Ty r Al a Pr o Gl y Gl u Thr Val Lys 65 70 75 80 I l e Gl y Cy s As n Thr Cy s Val Cy s Ar g As p Ar g Lys Tr p As n Cy s Thr 85 90 95 As p Hi s Val Cy s As p Al a Thr Cy s Ser Thr Ile Gl y Met Al a Hi s Ty r 100 105 110 Leu Thr Phe As p Gl y Leu Lys Ty r Leu Phe Pr o Gl y Gl u Cy s Gl n Ty r 115 120 125 Val Leu Val Gl n As p Ty r Cy s Gl y Ser As n Pr o Gl y Thr Phe Ar g I l e 130 135 140 Leu Val Gl y As n Lys Gl y Cy s Ser Hi s Pr o Ser Val Lys Cy s Lys Lys 145 150 155 160 Ar g Val Thr I l e Leu Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y 165 170 175

   Page 193

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   Gl u Val As n Val 180 Lys Ar g Pr o Met Lys 185 As p Gl u Thr Hi s Phe 190 Gl u Val Val Gl u Ser Gl y Ar g Ty r Ile Ile Leu Leu Leu Gl y Lys Al a Leu Ser 195 200 205 Val Val Tr p As p Ar g Hi s Leu Ser Ile Ser Val Val Leu Lys Gl n Thr 210 215 220 Ty r Gl n Gl u Lys Val Cy s Gl y Leu Cy s Gl y As n Phe As p Gl y I l e Gl n 225 230 235 240 As n As n As p Leu Thr Ser Ser As n Leu Gl n Val Gl u Gl u As p Pr o Val 245 250 255 As p Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cy s Al a As p Thr Ar g 260 265 270 Lys Val Pr o Leu As p Ser Ser Pr o Al a Thr Cy s Hi s As n As n I l e Met 275 280 285 Lys Gl n Thr Met Val As p Ser Ser Cy s Ar g Ile Leu Thr Ser As p Val 290 295 300 Phe Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu As p Val

   Page 194

   2018203206 08 May 2018

   97047_1

   305 310 315 320

   Cys I l e Ty r As p Thr 325 Cys Ser Cys Gl u Ser 330 I l e Gl y As p Cy s Al a 335 Al a Phe Cys As p Thr Ile Al a Al a Tyr Al a Hi s Val Cys Al a Gl n Hi s Gl y 340 345 350 Lys Val Val Thr Tr p Ar g Thr Al a Thr Leu Cys Pr o Gl n Ser Cys Gl u 355 360 365 Gl u Ar g As n Leu Ar g Gl u As n Gl y Tyr Gl u Al a Gl u Tr p Ar g Tyr As n 370 375 380 Ser Cys Al a Pr o Al a Cys Gl n Val Thr Cys Gl n Hi s Pr o Gl u Pr o Leu 385 390 395 400 Al a Cys Pr o Val Gl n Cys Val Gl u Gl y Cys Hi s Al a Hi s Cys Pr o Pr o 405 410 415 Gl y Lys I l e Leu As p Gl u Leu Leu Gl n Thr Cys Val As p Pr o Gl u As p 420 425 430 Cys Pr o Val Cys Gl u Val Al a Gl y Ar g Ar g Phe Al a Ser Gl y Lys Lys 435 440 445

   Page 195

   97047_1

   2018203206 08 May 2018

   Val Thr Leu As n Pr o Ser As p Pr o Gl u Hi s Cy s Gl n I l e Cy s Hi s Cy s 450 455 460 As p Val Val As n Leu Thr Cy s Gl u Al a Cy s Gl n Gl u Pr o I l e Ser Gl y 465 470 475 480 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 485 490 495 Thr Ser Gl y Ser Gl u Thr Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u 500 505 510 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 515 52 0 525 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u 530 535 540 Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Ser Pr o Al a Gl y Ser Pr o Thr Ser 545 550 555 560 Thr Gl u Gl u Gl y Thr Ser Gl u Se r Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 565 570 575 Ser Gl u Pr o Al a Thr Ser Gl y Se r Gl u Thr Pr o Gl y Thr Ser Gl u Ser

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   580 585 97047_1 590 Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 595 600 605 Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y 610 615 620 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser 625 630 635 640 Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 645 650 655 Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 660 665 670 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a 675 680 685 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 690 695 700 Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 705 710 715 720

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   Thr Ser Thr Gl u Pr o 725 Ser Gl u Gl y Ser Al a 730 Pr o Gl y Ser Gl u Pr o 735 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 740 745 750 Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o As p 755 760 765 Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser 770 775 780 Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Leu Val Pr o Ar g Gl y Ser 785 790 795 800 Gl y Gl y Al a Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu 805 810 815 Ser Tr p As p Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a 820 825 830 Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val 835 840 845 Val Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n

   Page 198

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   97047_1

   850 855 860

   I l e 865 Al a Lys Pr o Ar g Pr o 870 Pr o Tr p Met Gl y Leu 875 Leu Gl y Pr o Thr I l e 880 Gl n Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a 885 890 895 Ser Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Ty r Tr p Lys Al a 900 905 910 Ser Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u 915 920 925 Asp As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Ty r Val Tr p Gl n Val 930 935 940 Leu Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o Leu Cy s Leu Thr Ty r 945 950 955 960 Ser Tyr Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu 965 970 975 I l e Gly Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys 980 985 990

   Page 199

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   Thr Gl n Thr Leu Hi s Lys Phe I l e Leu Leu Phe Al a Val Phe As p Gl u 995 1000 1005 Gl y Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p 1010 1015 1020 Ar g As p Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val 1025 1030 1035 As n Gl y Tyr Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cy s Hi s 1040 1045 1050 Ar g Lys Ser Val Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o 1055 1060 1065 Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g 1070 1075 1080 As n Hi s Ar g Gl n Al a Ser Leu Gl u Ile Ser Pr o Ile Thr Phe Leu 1085 1090 1095 Thr Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu Leu Phe 1100 1105 1110

   Cys Hi s

   I l e Ser Ser

   Hi s Gl n

   Hi s Asp Gl y Met Gl u

   Al a Tyr Val

   Page 200

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   1115 1120 97047 _1 1125 Lys Val As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n 1130 1135 1140 As n Gl u Gl u Al a Gl u As p Tyr As p As p As p Leu Thr As p Ser Gl u 1145 1150 1155 Met As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser Phe I l e 1160 1165 1170 Gl n I l e Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s 1175 1180 1185 Tyr I l e Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o Leu Val 1190 1195 1200 Leu Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n As n 1205 1210 1215 Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met 1220 1225 1230 Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a I l e Gl n Hi s 1235 1240 1245

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   Gl u Ser 1250 Gl y Ile Leu Gl y Pr o 1255 Leu Leu Ty r Gl y Gl u 1260 Val Gl y As p Thr Leu Leu Ile Ile Phe Lys As n Gl n Al a Ser Ar g Pr o Ty r As n 1265 1270 1275 I l e Ty r Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Ty r Ser Ar g 1280 1285 1290 Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o I l e Leu 1295 1300 1305 Pr o Gl y Gl u Ile Phe Lys Ty r Lys Tr p Thr Val Thr Val Gl u As p 1310 1315 1320 Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s Leu Thr Ar g Ty r Ty r Ser 1325 1330 1335 Ser Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y 1340 1345 1350 Pr o Leu Leu Ile Cy s Ty r Lys Gl u Ser Val As p Gl n Ar g Gl y As n 1355 1360 1365 Gl n I l e Met Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe

   Page 202

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   97047_1

   1370 1375 1380

   As p Gl u As n Ar g Ser Tr ^p Ty r Leu Thr Gl u As n Ile Gl n Ar g Phe 1385 1390 1395 Leu Pr o As n Pr o Al a Gl ^y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n 1400 1405 1410 Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val Phe As p Ser 1415 1420 1425 Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p Ty r I l e 1430 1435 1440 Leu Ser I l e Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser 1445 1450 1455 Gl y Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr 1460 1465 1470 Leu Phe Pr o Phe Ser Gl ^y Gl u Thr Val Phe Met Ser Met Gl u As n 1475 1480 1485 Pr o Gl y Leu Tr p Ile Le u Gl y Cy s Hi s As n Ser As p Phe Ar g As n

   1490 1495 1500

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   Ar g Gl y Met Thr Al a Leu Leu 1510 Lys Val Ser Ser Cy s 1515 As p Lys As n 1505 Thr Gl y As p Ty r Ty r Gl u As p Ser Ty r Gl u As p Ile Ser Al a Ty r 1520 1525 1530 Leu Leu Ser Lys As n As n Al a Ile Gl u Pr o Ar g Ser Phe Ser Gl n 1535 1540 1545 As n Pr o Pr o Val Leu Lys Ar g Hi s Gl n Ar g Gl u Ile Thr Ar g Thr 1550 1555 1560 Thr Leu Gl n Ser As p Gl n Gl u Gl u Ile As p Ty r As p As p Thr I l e 1565 1570 1575 Ser Val Gl u Met Lys Lys Gl u As p Phe As p Ile Ty r As p Gl u As p 1580 1585 1590 Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Ty r 1595 1600 1605 Phe I l e Al a Al a Val Gl u Ar g Leu Tr p As p Ty r Gl y Met Ser Ser 1610 1615 1620 Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o

   Page 204

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   1625 1630 97047 _1 1635 Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe 1640 1645 1650 Thr Gl n Pr o Leu Ty r Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu 1655 1660 1665 Leu Gl y Pr o Ty r Ile Ar g Al a Gl u Val Gl u As p As n I l e Met Val 1670 1675 1680 Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Ty r Ser Phe Ty r Ser Ser 1685 1690 1695 Leu I l e Ser Ty r Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g 1700 1705 1710 Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr Ty r Phe Tr p Lys 1715 1720 1725 Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u Phe As p Cy s Lys 1730 1735 1740 Al a Tr p Al a Ty r Phe Ser As p Val As p Leu Gl u Lys As p Val Hi s 1745 1750 1755

   Page 205

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   Ser Gl y 1760 Leu Ile Gl y Pr o Leu 1765 Leu Val Cy s Hi s Thr 1770 As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu 1775 1780 1785 Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p Ty r Phe Thr Gl u 1790 1795 1800 As n Met Gl u Ar g As n Cy s Ar g Al a Pr o Cy s As n Ile Gl n Met Gl u 1805 1810 1815 As p Pr o Thr Phe Lys Gl u As n Ty r Ar g Phe Hi s Al a I l e As n Gl y 1820 1825 1830 Ty r I l e Met As p Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n 1835 1840 1845 Ar g I l e Ar g Tr p Ty r Leu Leu Ser Met Gl y Ser As n Gl u As n I l e 1850 1855 1860 Hi s Ser I l e Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys 1865 1870 1875 Gl u Gl u Ty r Lys Met Al a Leu Ty r As n Leu Ty r Pr o Gl y Val Phe

   Page 206

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   1880 1885 97047 _1 1890 Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val 1895 1900 1905 Gl u Cys Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu 1910 1915 1920 Phe Leu Val Tyr Ser As n Lys Cys Gl n Thr Pr o Leu Gl y Met Al a 1925 1930 1935 Ser Gl y Hi s Ile Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Tyr 1940 1945 1950 Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser 1955 1960 1965 I l e As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p I l e Lys Val 1970 1975 1980 As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y Ile Lys Thr Gl n Gl y 1985 1990 1995 Al a Ar g Gl n Lys Phe Ser Ser Leu Tyr Ile Ser Gl n Phe I l e I l e 2000 2005 2010

   Page 207

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   Met Tyr 2015 Ser Leu As p Gl y Lys 2020 Lys Tr p Gl n Thr Tyr 2025 Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n Val As p Ser Ser 2030 2035 2040 Gl y I l e Lys Hi s As n Ile Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr 2045 2050 2055 I l e Ar g Leu Hi s Pr o Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g 2060 2065 2070 Met Gl u Leu Met Gl y Cys As p Leu As n Ser Cys Ser Met Pr o Leu 2075 2080 2085 Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n Ile Thr Al a Ser 2090 2095 2100 Ser Tyr Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a 2105 2110 2115 Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val 2120 2125 2130 As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met

   Page 208

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   2135 Thr 2140 Gl n Gl y Val Lys 2145 Ser 2160 Leu Leu Thr Lys Val 2150 Gl y Val Thr Thr 2155 Ser Met Tyr Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y 2165 2170 2175 Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val Phe 2180 2185 2190 Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu As p 2195 2200 2205 Pr o Pr o Leu Leu Thr Ar g Tyr Leu Ar g Ile Hi s Pr o Gl n Ser Tr p 2210 2215 2220 Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu Gl y Cys Gl u Al a 2225 2230 2235

   Gl n Asp Leu Tyr 2240

   <210> 103 <211> 1959 <212> PRT

   Page 209

   2018203206 08 May 2018

   97047_1 <213> Artificial Sequence <220>

   <223> pSYN- FVI I I - 169 mat ur e Pr ot ei n sequence <400> 103

   Al a 1 Thr Ar g Ar g Tyr 5 Tyr Leu Gl y Al a Val 10 Gl u Leu Ser Tr p As p 15 Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g Phe Pr o Pr o 20 25 30 Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e Al a Lys Pr o 50 55 60 Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr Ile Gl n Al a Gl u Val 65 70 75 80 Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser Hi s Pr o Val 85 90 95 Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a 100 105 110

   Page 210

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   Gl u Ty r As p As p Gl n 115 Thr Ser Gl n Ar g Gl u 120 Lys Gl u As p As p 125 Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Ty r Val Tr p Gl n Val Leu Lys Gl u As n 130 135 140 Gl y Pr o Met Al a Ser As p Pr o Leu Cy s Leu Thr Ty r Ser Ty r Leu Ser 145 150 155 160 Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e Gl y Al a Leu 165 170 175 Leu Val Cy s Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr Leu 180 185 190 Hi s Lys Phe I l e Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser Tr p 195 200 205 Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a Ser 210 215 220 Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Ty r Val As n Ar g 225 230 235 240 Ser Leu Pr o Gl y Leu Ile Gl y Cy s Hi s Ar g Lys Ser Val Ty r Tr p Hi s

   Page 211

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   97047_1

   245 250 255

   Val I l e Gl y Met 260 Gl y Thr Thr Pr o Gl u 265 Val Hi s Ser I l e Phe 270 Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u I l e 275 280 285 Ser Pr o I l e Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met As p Leu Gl y 290 295 300 Gl n Phe Leu Leu Phe Cy s Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y Met 305 310 315 320 Gl u Al a Ty r Val Lys Val As p Ser Cy s Pr o Gl u Gl u Pr o Gl n Leu Ar g 325 330 335 Met Lys As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p Leu Thr As p 340 345 350 Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser Phe 355 360 365 I l e Gl n I l e Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s 370 375 380

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   Tyr 385 I l e Al a Al a Gl u Gl u 390 Gl u As p Tr p Asp Tyr 395 Al a Pr o Leu Val Leu 400 Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n As n Gl y Pr o 405 410 415 Gl n Ar g I l e Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met Al a Tyr Thr 420 425 430 As p Gl u Thr Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y I l e 435 440 445 Leu Gl y Pr o Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu Leu I l e I l e 450 455 460 Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n Ile Tyr Pr o Hi s Gl y I l e 465 470 475 480 Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys 485 490 495 Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u Ile Phe Lys Tyr Lys 500 505 510 Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g Cys

   Page 213

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   97047_1

   515 520 525

   Leu Thr 530 Arg Tyr Tyr Ser Ser 535 Phe Val As n Met Gl u 540 Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu Leu Ile Cys Tyr Lys Gl u Ser Val As p 545 550 555 560 Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val I l e Leu Phe 565 570 575 Ser Val Phe As p Gl u As n Ar g Ser Tr p Tyr Leu Thr Gl u As n I l e Gl n 580 585 590 Arg Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe 595 600 605 Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Tyr Val Phe As p Ser 610 615 620 Leu Gl n Leu Ser Val Cys Leu Hi s Gl u Val Al a Tyr Tr p Tyr I l e Leu 625 630 635 640 Ser I l e Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y Tyr 645 650 655

   Page 214

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   Thr Phe Lys Hi s Lys Met Val Tyr Gl u As p Thr Leu Thr Leu Phe Pr o 660 665 670 Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu Tr ^p 675 680 685 I l e Leu Gl y Cys Hi s As n Ser As p Phe Ar g As n Ar g Gl y Met Thr Al a 690 695 700 Leu Leu Lys Val Ser Ser Cys As p Lys As n Thr Gl y As p Tyr Tyr Gl u 705 710 715 72 0 As p Ser Tyr Gl u As p Ile Ser Al a Tyr Leu Leu Ser Lys As n As n Al a 725 730 735 I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Gl y Al a Pr o Gl y Thr Ser Gl u 740 745 750 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl ^y 755 760 765 Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 770 775 780 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u

   Page 215

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   97047_1

   785 790 795 800

   Se r Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u 805 810 815 Gl ^y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 820 825 830 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 835 840 845 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 850 855 860 Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 86 5 870 875 880 Gl ^y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr 885 890 895 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 900 905 910 Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 915 920 925

   Page 216

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   Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 930 935 940 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl ^y Ser Gl u Pr o Al a Thr Ser Gl y 94 5 950 955 960 Se r Gl u Thr Pr o Gl y Ser Pr o Al a Gl ^y Ser Pr o Thr Ser Thr Gl u Gl u 965 970 975 Gl ^y Thr Ser Thr Gl u Pr o Ser Gl u Gl ^y Ser Al a Pr o Gl y Thr Ser Thr 980 98 5 990 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y 995 1000 100 5

   Ser Gl u

   Thr Pr o Gl y Thr Ser

   Gl u Ser Al a Thr Pr o

   Gl u Ser Gl y

   1010

   1015

   1020

   Pr o Gl y

   Thr Ser Thr

   Gl u Pr o

   Ser Gl u Gl y Ser Al a

   Pr o Al a Ser

   1025

   1030

   1035

   Ser Pr o

   Pr o Val Leu Lys Ar g

   Hi s Gl n Al a Gl u

   Ile

   Thr Ar g Thr

   1040

   1045

   1050

   Thr Leu

   Gl n Ser Asp Gl n Gl u

   Gl u I l e As p Ty r As p

   As p Thr I l e

   Page 217

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   97047_1

   1055 1060 1065

   Ser Val 1070 Gl u Met Lys Lys Gl u 1075 As p Phe As p Ile Ty r 1080 As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Ty r 1085 1090 1095 Phe I l e Al a Al a Val Gl u Ar g Leu Tr p As p Ty r Gl y Met Ser Ser 1100 1105 1110 Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o 1115 1120 1125 Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe 1130 1135 1140 Thr Gl n Pr o Leu Ty r Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu 1145 1150 1155 Leu Gl y Pr o Ty r Ile Ar g Al a Gl u Val Gl u As p As n I l e Met Val 1160 1165 1170

   Thr Phe

   Arg Asn Gl n Al a Ser

   Arg Pro Tyr Ser Phe

   Tyr Ser Ser

   1175

   1180

   1185

   Page 218

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   Leu I l e 1190 Ser Tyr Gl u Gl u As p 1195 Gl n Ar g Gl n Gl y Al a 1200 Gl u Pr o Ar g Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys 1205 1210 1215 Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys 1220 1225 1230 Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u Lys As p Val Hi s 1235 1240 1245 Ser Gl y Leu Ile Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu 1250 1255 1260 As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu 1265 1270 1275 Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u 1280 1285 1290 As n Met Gl u Ar g As n Cys Ar g Al a Pr o Cys As n Ile Gl n Met Gl u 1295 1300 1305 As p Pr o Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s Al a I l e As n Gl y

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   1310 1315 97047 _1 1320 Ty r I l e Met As p Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n 1325 1330 1335 Ar g I l e Ar g Tr p Ty r Leu Leu Ser Met Gl y Ser As n Gl u As n I l e 1340 1345 1350 Hi s Ser I l e Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys 1355 1360 1365 Gl u Gl u Ty r Lys Met Al a Leu Ty r As n Leu Ty r Pr o Gl y Val Phe 1370 1375 1380 Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val 1385 1390 1395 Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu 1400 1405 1410 Phe Leu Val Ty r Ser Asn Lys Cy s Gl n Thr Pr o Leu Gl y Met Al a 1415 1420 1425 Ser Gl y Hi s Ile Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Ty r 1430 1435 1440

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   Gl y Gl n 1445 Tr p Al a Pr o Lys Leu 1450 Al a Ar g Leu Hi s Ty r 1455 Ser Gl y Ser I l e As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p I l e Lys Val 1460 1465 1470 As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y Ile Lys Thr Gl n Gl y 1475 1480 1485 Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r Ile Ser Gl n Phe I l e I l e 1490 1495 1500 Met Ty r Ser Leu As p Gl y Lys Lys Tr p Gl n Thr Ty r Ar g Gl y As n 1505 1510 1515 Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n Val As p Ser Ser 1520 1525 1530 Gl y I l e Lys Hi s As n Ile Phe As n Pr o Pr o Ile Ile Al a Ar g Ty r 1535 1540 1545 I l e Ar g Leu Hi s Pr o Thr Hi s Ty r Ser Ile Ar g Ser Thr Leu Ar g 1550 1555 1560 Met Gl u Leu Met Gl y Cy s As p Leu As n Ser Cy s Ser Met Pr o Leu

   Page 221

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   1565 1570 97047 _1 1575 Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n Ile Thr Al a Ser 1580 1585 1590 Ser Ty r Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a 1595 1600 1605 Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val 1610 1615 1620 As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met 1625 1630 1635 Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr 1640 1645 1650 Ser Met Ty r Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y 1655 1660 1665 Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val Phe 1670 1675 1680 Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu As p 1685 1690 1695

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   97047_1

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   Pr o Pr o 1700 Leu Leu Thr Ar g Ty r 1705 Leu Ar g Ile Hi s Pr o 1710 Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a 1715 1720 1725 Gl n As p Leu Ty r As p Lys Thr Hi s Thr Cy s Pr o Pr o Cy s Pr o Al a 1730 1735 1740 Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys 1745 1750 1755 Pr o Lys As p Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cy s 1760 1765 1770 Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n 1775 1780 1785 Tr p Ty r Val As p Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o 1790 1795 1800 Ar g Gl u Gl u Gl n Ty r As n Ser Thr Ty r Ar g Val Val Ser Val Leu 1805 1810 1815 Thr Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Ty r Lys Cy s

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   97047_1

   1820 1825 1830

   Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr I l e 1835 1840 1845 Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Ty r Thr Leu 1850 1855 1860 Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr 1865 1870 1875 Cy s Leu Val Lys Gl y Phe Ty r Pr o Ser As p Ile Al a Val Gl u Tr p 1880 1885 1890 Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Ty r Lys Thr Thr Pr o Pr o 1895 1900 1905 Val Leu As p Ser As p Gl y Ser Phe Phe Leu Ty r Ser Lys Leu Thr 1910 1915 1920 Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser Cy s Ser 1925 1930 1935

   Val Met

   Hi s Gl u Al a Leu

   Hi s

   As n Hi s Ty r Thr Gl n

   Lys Ser Leu

   1940

   1945

   1950

   Page 224

   97047_1

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   Ser Leu Ser Pr o Gl y Lys 1955 <210> 104 <211> 1959 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN- FVI I I - 173 mat ur e Pr ot ei n <400> 104

   Al a Thr 1 Ar g Ar g Tyr 5 Tyr Leu Gl y Al a Val 10 Gl u Leu Ser Tr p As p 15 Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g Phe Pr o Pr o 20 25 30 Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val Tyr Lys Lys 35 40 45 Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e Al a Lys Pr o 50 55 60 Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr Ile Gl n Al a Gl u Val 65 70 75 80

   Page 225

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   Tyr As p Thr Val Val 85 I l e Thr Leu Lys As n 90 Met Al a Ser Hi s Pr o 95 Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a 100 105 110 Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys Val 115 120 125 Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu Lys Gl u As n 130 135 140 Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser Tyr Leu Ser 145 150 155 160 Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e Gl y Al a Leu 165 170 175 Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr Leu 180 185 190 Hi s Lys Phe I l e Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser Tr p 195 200 205 Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a Ser

   Page 226

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   97047_1

   210 215 220

   Al a Ar g Al a 225 Tr p Pr o Lys 230 Met Hi s Thr Val As n 235 Gl y Tyr Val As n Ar g 240 Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val Tyr Tr p Hi s 245 250 255 Val I l e Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e Phe Leu Gl u 260 265 270 Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u I l e 275 280 285 Ser Pr o I l e Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met As p Leu Gl y 290 295 300 Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y Met 305 310 315 320 Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu Ar g 325 330 335 Met Lys As n As n Gl u Gl u Al a Gl u As p Tyr As p As p As p Leu Thr As p 340 345 350

   Page 227

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   Ser Gl u Met 355 As p Val Val Ar g Phe Asp Asp Asp Asn Ser Pr o Ser Phe 360 365 I l e Gl n I l e Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s 370 375 380 Tyr I l e Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o Leu Val Leu 385 390 395 400 Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n As n Gl y Pr o 405 410 415 Gl n Ar g I l e Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met Al a Tyr Thr 420 425 430 As p Gl u Thr Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y I l e 435 440 445 Leu Gl y Pr o Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu Leu I l e I l e 450 455 460 Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n Ile Tyr Pr o Hi s Gl y I l e 465 470 475 480 Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys

   Page 228

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   97047_1

   485 490 495

   Hi s Leu Lys As p Phe 500 Pr o Ile Leu Pr o 505 Gl y Gl u I l e Phe Lys 510 Ty r Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s 515 520 525 Leu Thr Ar g Ty r Ty r Ser Ser Phe Val As n Met Gl u Ar g As p Leu Al a 530 535 540 Ser Gl y Leu I l e Gl y Pr o Leu Leu Ile Cy s Ty r Lys Gl u Ser Val As p 545 550 555 560 Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val I l e Leu Phe 565 570 575 Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u As n I l e Gl n 580 585 590 Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe 595 600 605 Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val Phe As p Ser 610 615 620

   Page 229

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   Leu 625 Gl n Leu Ser Val Cy s 630 Leu Hi s Gl u Val Al a 635 Ty r Tr p Ty r I l e Leu 640 Ser I l e Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y Ty r 645 650 655 Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr Leu Phe Pr o 660 665 670 Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu Tr p 675 680 685 I l e Leu Gl y Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y Met Thr Al a 690 695 700 Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p Ty r Ty r Gl u 705 710 715 720 As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys As n As n Al a 725 730 735 I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Gl y Al a Pr o Gl y Thr Ser Gl u 740 745 750 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y

   Page 230

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   97047_1

   755 760 765

   Se r Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 770 775 780 Gl ^y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u 78 5 790 795 800 Se r Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u 805 810 815 Gl ^y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 820 825 830 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 835 840 845 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 850 855 860 Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 86 5 870 875 880 Gl ^y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr 885 890 895

   Page 231

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   Gl u Pr o Ser Gl u 900 Gl y Ser Al a Pr o Gl y Thr 905 Ser Gl u Ser Al a 910 Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 915 920 925 Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 930 935 940 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y 945 950 955 960 Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 965 970 975 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 980 985 990 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y 995 1000 1005

   Ser Gl u

   Thr Pr o Gl y Thr Ser

   Gl u Ser Al a Thr Pr o

   Gl u Ser Gl y

   1010

   1015

   1020

   Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Al a Ser

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   97047_1

   1025 1030 1035

   Ser Pr o 1040 Pr o Val Leu Lys Ar g 1045 Hi s Gl n Ar g Gl u Ile 1050 Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u Ile As p Tyr As p As p Thr I l e 1055 1060 1065 Ser Val Gl u Met Lys Lys Gl u As p Phe As p Ile Tyr As p Gl u As p 1070 1075 1080 Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Tyr 1085 1090 1095 Phe I l e Al a Al a Val Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser 1100 1105 1110 Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o 1115 1120 1125 Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe 1130 1135 1140 Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu

   1145 1150 1155

   Page 233

   97047_1

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   Leu Gl y Pr o Tyr I l e Ar g Al a 1165 Gl u Val Gl u As p As n 1170 I l e Met Val 1160 Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser Ser 1175 1180 1185 Leu I l e Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g 1190 1195 1200 Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys 1205 1210 1215 Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys 1220 1225 1230 Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u Lys As p Val Hi s 1235 1240 1245 Ser Gl y Leu Ile Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu 1250 1255 1260 As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu 1265 1270 1275 Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u

   Page 234

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   97047_1

   1280 1285 1290

   As n Met 1295 Gl u Ar g As n Cy s Ar g 1300 Al a Pr o Cy s As n Ile 1305 Gl n Met Gl u As p Pr o Thr Phe Lys Gl u As n Ty r Ar g Phe Hi s Al a I l e As n Gl y 1310 1315 1320 Ty r I l e Met As p Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n 1325 1330 1335 Ar g I l e Ar g Tr p Ty r Leu Leu Ser Met Gl y Ser As n Gl u As n I l e 1340 1345 1350 Hi s Ser I l e Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys 1355 1360 1365 Gl u Gl u Ty r Lys Met Al a Leu Ty r As n Leu Ty r Pr o Gl y Val Phe 1370 1375 1380 Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val 1385 1390 1395 Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu

   1400 1405 1410

   Page 235

   97047 1

   2018203206 08 May 2018

   Phe Leu 1415 Val Tyr Ser As n Lys 1420 Cys Gl n Thr Pr o Leu 1425 Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Tyr 1430 1435 1440 Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser 1445 1450 1455 I l e As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p I l e Lys Val 1460 1465 1470 As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y Ile Lys Thr Gl n Gl y 1475 1480 1485 Al a Ar g Gl n Lys Phe Ser Ser Leu Tyr Ile Ser Gl n Phe I l e I l e 1490 1495 1500 Met Tyr Ser Leu As p Gl y Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n 1505 1510 1515 Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n Val As p Ser Ser 1520 1525 1530 Gl y I l e Lys Hi s As n Ile Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr

   Page 236

   2018203206 08 May 2018

   1535 1540 97047 _1 1545 I l e Ar g Leu Hi s Pr o Thr Hi s Ty r Ser Ile Ar g Ser Thr Leu Ar g 1550 1555 1560 Met Gl u Leu Met Gl y Cy s As p Leu As n Ser Cy s Ser Met Pr o Leu 1565 1570 1575 Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n Ile Thr Al a Ser 1580 1585 1590 Ser Ty r Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a 1595 1600 1605 Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val 1610 1615 1620 As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met 1625 1630 1635 Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr 1640 1645 1650 Ser Met Ty r Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y 1655 1660 1665

   Page 237

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   Hi s Gl n 1670 Tr p Thr Leu Phe Phe 1675 Gl n As n Gl y Lys Val 1680 Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu As p 1685 1690 1695 Pr o Pr o Leu Leu Thr Ar g Ty r Leu Ar g Ile Hi s Pr o Gl n Ser Tr p 1700 1705 1710 Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a 1715 1720 1725 Gl n As p Leu Ty r As p Lys Thr Hi s Thr Cy s Pr o Pr o Cy s Pr o Al a 1730 1735 1740 Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys 1745 1750 1755 Pr o Lys As p Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cy s 1760 1765 1770 Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n 1775 1780 1785 Tr p Ty r Val As p Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o

   Page 238

   2018203206 08 May 2018

   97047_1

   1790 1795 1800

   Ar g Gl u 1805 Gl u Gl n Ty r As n Ser 1810 Thr Ty r Ar g Val Val 1815 Ser Val Leu Thr Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Ty r Lys Cy s 1820 1825 1830 Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr I l e 1835 1840 1845 Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Ty r Thr Leu 1850 1855 1860 Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr 1865 1870 1875 Cy s Leu Val Lys Gl y Phe Ty r Pr o Ser As p Ile Al a Val Gl u Tr p 1880 1885 1890 Gl u Ser Asn Gl y Gl n Pr o Gl u As n As n Ty r Lys Thr Thr Pr o Pr o 1895 1900 1905 Val Leu As p Ser As p Gl y Ser Phe Phe Leu Ty r Ser Lys Leu Thr

   1910 1915 1920

   Page 239

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   Val As p 1925 Lys Ser Ar g Tr p Gl n 1930 Gl n Gl y As n Val Phe 1935 Ser Cys Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu 1940 1945 1950 Ser Leu Ser Pr o Gl y Lys

   1955 <210> 105 <211> 1984 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> FVI I I 195 pr ot ei n sequence <400> 105

   Met 1 Gl n I l e Gl u Leu Ser 5 Thr Cys Phe Phe 10 Leu Cys Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g 35 40 45

   Page 240

   97047_1

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   Phe Pr o 50 Pr o Ar g Val Pr o Lys 55 Ser Phe Pr o Phe As n 60 Thr Ser Val Val Ty r Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e 65 70 75 80 Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr I l e Gl n 85 90 95 Al a Gl u Val Ty r As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser 100 105 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Ty r Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Ty r As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Ty r Val Tr p Gl n Val Leu 145 150 155 160 Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o Leu Cy s Leu Thr Ty r Ser 165 170 175 Ty r Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e

   Page 241

   2018203206 08 May 2018

   180 185 97047_1 190 Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205 Gl n Thr Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y 210 215 220 Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p 225 230 235 240 Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr 245 250 255 Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val 260 265 270 Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285 Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser 290 295 300 Leu Gl u I l e Ser Pr o Ile Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met 305 310 315 320

   Page 242

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   As p Leu Gl y Gl n Phe 325 Leu Leu Phe Cy s Hi s 330 Ile Ser Ser Hi s Gl n 335 Hi s As p Gl y Met Gl u Al a Ty r Val Lys Val As p Ser Cy s Pr o Gl u Gl u Pr o 340 345 350 Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p 355 360 365 Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser 370 375 380 Pr o Ser Phe I l e Gl n Ile Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr 385 390 395 400 Tr p Val Hi s Ty r Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Ty r Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p As p Ar g Ser Ty r Lys Ser Gl n Ty r Leu As n 420 425 430 As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Ty r Lys Lys Val Ar g Phe Met 435 440 445 Al a Ty r Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a I l e Gl n Hi s Gl u

   Page 243

   2018203206 08 May 2018

   97047_1

   450 455 460

   Ser 465 Gl y I l e Leu Gl y Pr o 470 Leu Leu Tyr Gl y Gl u 475 Val Gl y As p Thr Leu 480 Leu I l e I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n I l e Tyr Pr o 485 490 495 Hi s Gl y I l e Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys 500 505 510 Gl y Val Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe 515 520 525 Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p 530 535 540 Pr o Ar g Cys Leu Thr Ar g Tyr Tyr Ser Ser Phe Val As n Met Gl u Ar g 545 550 555 560 As p Leu Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cys Tyr Lys Gl u 565 570 575 Ser Val As p Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val 580 585 590

   Page 244

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   2018203206 08 May 2018

   I l e Leu Phe Ser 595 Val Phe As p Gl u As n 600 Ar g Ser Tr p Tyr 605 Leu Thr Gl u As n I l e Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p 610 615 620 Pr o Gl u Phe Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Tyr Val 625 630 635 640 Phe As p Ser Leu Gl n Leu Ser Val Cys Leu Hi s Gl u Val Al a Tyr Tr p 645 650 655 Tyr I l e Leu Ser Ile Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe 660 665 670 Ser Gl y Tyr Thr Phe Lys Hi s Lys Met Val Tyr Gl u As p Thr Leu Thr 675 680 685 Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o 690 695 700 Gl y Leu Tr p I l e Leu Gl y Cys Hi s As n Ser As p Phe Ar g As n Ar g Gl y 705 710 715 720 Met Thr Al a Leu Leu Lys Val Ser Ser Cys As p Lys As n Thr Gl y As p

   Page 245

   2018203206 08 May 2018

   97047_1

   725 730 735

   Tyr Tyr Gl u Asp Ser 740 Tyr Gl u As p Ile 745 Ser Al a Tyr Leu Leu 750 Ser Lys As n As n Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Pr o Pr o Val Leu 755 760 765 Lys Ar g Hi s Gl n Ar g Gl u Ile Thr Ar g Thr Thr Leu Gl n Gl y Al a Pr o 770 775 780 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o 785 790 795 800 Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 805 810 815 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 820 825 830 Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Thr Pr o Gl y 835 840 845 Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 850 855 860

   Page 246

   97047 1

   2018203206 08 May 2018

   Thr 865 Gl y Ser Pr o Gl y Al a 870 Ser Pr o Gl y Thr Ser 875 Ser Thr Gl y Ser Pr o 880 Gly Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Thr 885 890 895 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 900 905 910 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 915 920 925 Al a Ser Ser Ser Asp Gl n Gl u Gl u I l e As p Tyr As p As p Thr I l e Ser 930 935 940 Val Gl u Met Lys Lys Gl u As p Phe As p Ile Tyr As p Gl u As p Gl u As n 945 950 955 960 Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Tyr Phe I l e Al a 965 970 975 Al a Val Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser Ser Pr o Hi s Val 980 985 990 Leu Ar g Asn Ar g Al a Gl n Ser Gl y Ser Val Pr o Gl n Phe Lys Lys Val

   Page 247

   2018203206 08 May 2018

   97047_1

   995 1000 1005

   Val Phe 1010 Gl n Gl u Phe Thr As p 1015 Gl y Ser Phe Thr Gl n 1020 Pr o Leu Ty r Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu Leu Gl y Pr o Ty r I l e 1025 1030 1035 Ar g Al a Gl u Val Gl u As p As n Ile Met Val Thr Phe Ar g As n Gl n 1040 1045 1050 Al a Ser Ar g Pr o Ty r Ser Phe Ty r Ser Ser Leu Ile Ser Ty r Gl u 1055 1060 1065 Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g Lys As n Phe Val Lys 1070 1075 1080 Pr o As n Gl u Thr Lys Thr Ty r Phe Tr p Lys Val Gl n Hi s Hi s Met 1085 1090 1095 Al a Pr o Thr Lys As p Gl u Phe As p Cy s Lys Al a Tr p Al a Ty r Phe 1100 1105 1110 Ser As p Val As p Leu Gl u Lys As p Val Hi s Ser Gl y Leu I l e Gl y 1115 1120 1125

   Page 248

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   Pr o Leu 1130 Leu Val Cy s Hi s Thr 1135 As n Thr Leu As n Pr o 1140 Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe 1145 1150 1155 As p Gl u Thr Lys Ser Tr p Ty r Phe Thr Gl u As n Met Gl u Ar g As n 1160 1165 1170 Cy s Ar g Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 1175 1180 1185 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr 1190 1195 1200 Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 1205 1210 1215 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 1220 1225 1230 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a 1235 1240 1245 Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser

   Page 249

   97047_1

   1260

   2018203206 08 May 2018

   1250 1255 Pr o Al a Gl y Ser Pr o Thr Ser 1265 1270 Ser Pr o Thr Ser Thr Gl u Gl u 1280 1285

   Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 1275

   Gl y Ser Pr o Al a Gl y Ser Pr o Thr 1290

   Ser Thr

   Gl u Gl u Gl y Thr Ser

   Gl u Ser Al a Thr Pr o

   Gl u Ser Gl y

   1295

   1300

   1305

   Pr o Gl y

   Thr Ser Thr

   Gl u Pr o

   Ser Gl u Gl y Ser Al a

   Pr o Gl y Al a

   1310

   1315

   1320

   Ser Ser

   Al a Pro Cys Asn I l e

   Gl n Met Gl u Asp Pr o

   Thr Phe Lys

   1325

   1330

   1335

   Gl u As n

   Tyr Arg Phe Hi s Al a

   I l e As n Gl y Ty r I l e

   Met Asp Thr

   1340

   1345

   1350

   Leu Pr o

   Gl y Leu Val Met Al a

   Gl n As p Gl n Ar g I l e

   Arg Trp Tyr

   1355

   1360

   1365

   Leu Leu

   Ser Met Gl y Ser Asn

   Gl u As n I l e Hi s

   Ser

   I l e Hi s Phe

   1370

   1375

   1380

   Page 250

   97047_1

   2018203206 08 May 2018

   Ser Gl y 1385 Hi s Val Phe Thr Val 1390 Ar g Lys Lys Gl u Gl u 1395 Tyr Lys Met Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe Gl u Thr Val Gl u Met 1400 1405 1410 Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys Leu I l e Gl y 1415 1420 1425 Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Tyr Ser 1430 1435 1440 As n Lys Cys Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g 1445 1450 1455 As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Tyr Gl y Gl n Tr p Al a Pr o 1460 1465 1470 Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser Ile As n Al a Tr p Ser 1475 1480 1485 Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o 1490 1495 1500 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe

   Page 251

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   1505 1510 97047 _1 1515 Ser Ser Leu Ty r Ile Ser Gl n Phe Ile Ile Met Ty r Ser Leu As p 1520 1525 1530 Gl y Lys Lys Tr p Gl n Thr Ty r Ar g Gl y As n Ser Thr Gl y Thr Leu 1535 1540 1545 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n 1550 1555 1560 I l e Phe As n Pr o Pr o Ile Ile Al a Ar g Ty r Ile Ar g Leu Hi s Pr o 1565 1570 1575 Thr Hi s Ty r Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y 1580 1585 1590 Cy s As p Leu As n Ser Cy s Ser Met Pr o Leu Gl y Met Gl u Ser Lys 1595 1600 1605 Al a I l e Ser As p Al a Gl n Ile Thr Al a Ser Ser Ty r Phe Thr As n 1610 1615 1620 Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n 1625 1630 1635

   Page 252

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   Gl y Ar g 1640 Ser As n Al a Tr p Ar g 1645 Pr o Gl n Val As n As n 1650 Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1655 1660 1665 Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1670 1675 1680 Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu 1685 1690 1695 Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p 1700 1705 1710 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1715 1720 1725 Ar g Tyr Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n I l e Al a 1730 1735 1740 Leu Ar g Met Gl u Val Leu Gl y Cys Gl u Al a Gl n As p Leu Tyr As p 1745 1750 1755 Lys Thr Hi s Thr Cys Pr o Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y

   Page 253

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   1760 1765 97047 _1 1770 Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1775 1780 1785 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val 1790 1795 1800 Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Tyr Val As p Gl y 1805 1810 1815 Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr 1820 1825 1830 As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n 1835 1840 1845 As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys 1850 1855 1860 Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a Lys Gl y 1865 1870 1875 Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p 1880 1885 1890

   Page 254

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   97047 1

   Gl u Leu 1895 Thr Lys As n Gl n Val 1900 Ser Leu Thr Cy s Leu 1905 Val Lys Gl y Phe Ty r Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n 1910 1915 1920 Pr o Gl u As n As n Ty r Lys Thr Thr Pr o Pr o Val Leu As p Ser As p 1925 1930 1935 Gl y Ser Phe Phe Leu Ty r Ser Lys Leu Thr Val As p Lys Ser Ar g 1940 1945 1950 Tr p Gl n Gl n Gl y As n Val Phe Ser Cy s Ser Val Met Hi s Gl u Al a 1955 1960 1965 Leu Hi s As n Hi s Ty r Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y 1970 1975 1980

   Lys <210> 106 <211> 2134 <212> PRT <213> Art i f i ci al Sequence

   Page 255

   2018203206 08 May 2018

   97047_1 <220>

   <223> FVIII 196 protein sequence <400> 106

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cy s Phe Phe 10 Leu Cy s Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Ty r Ty r Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Ty r Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val Gl y Al a Pr o 35 40 45 Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o 50 55 60 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 65 70 75 80 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 85 90 95 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o 100 105 110 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser

   Page 256

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   97047_1

   115 120 125

   Thr Gl y 130 Ser Pr o Gl y Ser Ser 135 Pr o Ser Al a Ser Thr 140 Gl y Thr Gl y Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 145 150 155 160 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 165 170 175 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 180 185 190 Al a Ser Ser As p Al a Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o 195 200 205 Phe As n Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr 210 215 220 As p Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu 225 230 235 240

   Leu Gl y Pr o Thr

   I l e Gl n Al a Gl u

   Val

   Tyr Asp Thr Val

   Val

   I l e Thr

   245

   250

   255

   Page 257

   97047_1

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   Leu Lys As n Met 260 Al a Ser Hi s Pr o Val 265 Ser Leu Hi s Al a Val 270 Gl y Val Ser Ty r Tr p Lys Al a Ser Gl u Gl y Al a Gl u Ty r As p As p Gl n Thr Ser 275 280 285 Gl n Arg Gl u Lys Gl u As p As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr 290 295 300 Ty r Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o 305 310 315 320 Leu Cy s Leu Thr Ty r Ser Ty r Leu Ser Hi s Val As p Leu Val Lys As p 325 330 335 Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cy s Ar g Gl u Gl y Ser 340 345 350 Leu Al a Lys Gl u Lys Thr Gl n Thr Leu Hi s Lys Phe I l e Leu Leu Phe 355 360 365 Al a Val Phe As p Gl u Gl y Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser 370 375 380 Leu Met Gl n As p Ar g As p Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met

   Page 258

   2018203206 08 May 2018

   97047_1

   385 390 395 400

   Hi s Thr Val As n Gl y 405 Ty r Val As n Ar g Ser 410 Leu Pr o Gl y Leu I l e 415 Gl y Cy s Hi s Ar g Lys Ser Val Ty r Tr p Hi s Val Ile Gl y Met Gl y Thr Thr 420 425 430 Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g 435 440 445 As n Hi s Ar g Gl n Al a Ser Leu Gl u Ile Ser Pr o Ile Thr Phe Leu Thr 450 455 460 Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu Leu Phe Cy s Hi s 465 470 475 480 I l e Ser Ser Hi s Gl n Hi s As p Gl y Met Gl u Al a Ty r Val Lys Val As p 485 490 495 Ser Cy s Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a 500 505 510 Gl u As p Ty r As p As p As p Leu Thr As p Ser Gl u Met As p Val Val Ar g 515 520 525

   Page 259

   97047_1

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   Phe Asp Asp Asp Asn Ser 530 Pr o 535 Ser Phe Ile Gl n Ile 540 Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Ty r Ile Al a Al a Gl u Gl u Gl u 545 550 555 560 As p Tr p As p Ty r Al a Pr o Leu Val Leu Al a Pr o As p As p Ar g Ser Ty r 565 570 575 Lys Ser Gl n Ty r Leu As n As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Ty r 580 585 590 Lys Lys Val Ar g Phe Met Al a Ty r Thr As p Gl u Thr Phe Lys Thr Ar g 595 600 605 Gl u Al a I l e Gl n Hi s Gl u Ser Gl y Ile Leu Gl y Pr o Leu Leu Ty r Gl y 610 615 620 Gl u Val Gl y As p Thr Leu Leu Ile Ile Phe Lys As n Gl n Al a Ser Ar g 625 630 635 640 Pr o Ty r As n I l e Ty r Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Ty r 645 650 655 Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o I l e

   Page 260

   2018203206 08 May 2018

   660 665 97047_1 670 Leu Pr o Gl y Gl u Ile Phe Lys Ty r Lys Tr p Thr Val Thr Val Gl u As p 675 680 685 Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s Leu Thr Ar g Ty r Ty r Ser Ser 690 695 700 Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu 705 710 715 720 Leu I l e Cy s Ty r Lys Gl u Ser Val As p Gl n Ar g Gl y As n Gl n I l e Met 725 730 735 Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe As p Gl u As n Ar g 740 745 750 Ser Tr p Ty r Leu Thr Gl u As n Ile Gl n Ar g Phe Leu Pr o As n Pr o Al a 755 760 765 Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser As n I l e Met Hi s 770 775 780 Ser I l e As n Gl y Ty r Val Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu 785 790 795 800

   Page 261

   97047_1

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   Hi s Gl u Val Al a Tyr 805 Tr p Tyr Ile Leu Ser 810 I l e Gl y Al a Gl n Thr 815 As p Phe Leu Ser Val Phe Phe Ser Gl y Tyr Thr Phe Lys Hi s Lys Met Val 820 825 830 Tyr Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe 835 840 845 Met Ser Met Gl u As n Pr o Gl y Leu Tr p Ile Leu Gl y Cys Hi s As n Ser 850 855 860 As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cys 865 870 875 880 As p Lys As n Thr Gl y As p Tyr Tyr Gl u As p Ser Tyr Gl u As p I l e Ser 885 890 895 Al a Tyr Leu Leu Ser Lys As n As n Al a Ile Gl u Pr o Ar g Ser Phe Ser 900 905 910 Gl n As n Pr o Pr o Val Leu Lys Ar g Hi s Gl n Ar g Gl u I l e Thr Ar g Thr 915 920 925 Thr Leu Gl n Gl y Al a Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser

   Page 262

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   97047 1

   930 935 940 Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl ^y Al a 945 950 955 960 Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl ^y Thr 965 970 97 5 Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl ^y Thr 980 985 990 Gl y Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a 995 1000 100 5

   Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y 1010 1015 1020 Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 1025 1030 1035 Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser 1040 1045 1050 Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Al a 1055 1060 1065

   Page 263

   97047_1

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   Ser Pr o 1070 Gl y Thr Ser Ser Thr 1075 Gl y Ser Pr o Al a Ser 1080 Ser Ser As p Gl n Gl u Gl u Ile As p Tyr As p As p Thr Ile Ser Val Gl u Met Lys 1085 1090 1095 Lys Gl u As p Phe As p Ile Tyr As p Gl u As p Gl u As n Gl n Ser Pr o 1100 1105 1110 Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Tyr Phe Ile Al a Al a Val 1115 1120 1125 Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser Ser Pr o Hi s Val Leu 1130 1135 1140 Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o Gl n Phe Lys Lys Val 1145 1150 1155 Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe Thr Gl n Pr o Leu Tyr 1160 1165 1170 Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu Leu Gl y Pr o Tyr I l e 1175 1180 1185 Ar g Al a Gl u Val Gl u As p As n Ile Met Val Thr Phe Ar g As n Gl n

   Page 264

   2018203206 08 May 2018

   97047_1

   1190 1195 1200

   Al a Ser 1205 Ar g Pr o Tyr Ser Phe 1210 Tyr Ser Ser Leu Ile 1215 Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g Lys As n Phe Val Lys 1220 1225 1230 Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met 1235 1240 1245 Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys Al a Tr p Al a Tyr Phe 1250 1255 1260 Ser As p Val As p Leu Gl u Lys As p Val Hi s Ser Gl y Leu I l e Gl y 1265 1270 1275 Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y 1280 1285 1290 Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe 1295 1300 1305 As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u As n Met Gl u Ar g As n

   1310 1315 1320

   Page 265

   97047_1

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   Cys Ar g 1325 Gl y Al a Pr o Thr Ser 1330 Gl u Ser Al a Thr Pr o 1335 Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr 1340 1345 1350 Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 1355 1360 1365 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 1370 1375 1380 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a 1385 1390 1395 Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser 1400 1405 1410 Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 1415 1420 1425 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr 1430 1435 1440 Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y

   Page 266

   2018203206 08 May 2018

   97047_1

   1445 1450 1455

   Pr o Gl y 1460 Thr Ser Thr Gl u Pr o 1465 Ser Gl u Gl y Ser Al a 1470 Pr o Gl y Al a Ser Ser Al a Pr o Cys As n Ile Gl n Met Gl u As p Pr o Thr Phe Lys 1475 1480 1485 Gl u As n Tyr Ar g Phe Hi s Al a Ile As n Gl y Tyr Ile Met As p Thr 1490 1495 1500 Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr 1505 1510 1515 Leu Leu Ser Met Gl y Ser As n Gl u As n Ile Hi s Ser I l e Hi s Phe 1520 1525 1530 Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Tyr Lys Met 1535 1540 1545 Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe Gl u Thr Val Gl u Met 1550 1555 1560 Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys Leu I l e Gl y

   1565 1570 1575

   Page 267

   97047_1

   2018203206 08 May 2018

   Gl u Hi s 1580 Leu Hi s Al a Gl y Met 1585 Ser Thr Leu Phe Leu 1590 Val Ty r Ser As n Lys Cy s Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g 1595 1600 1605 As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Ty r Gl y Gl n Tr p Al a Pr o 1610 1615 1620 Lys Leu Al a Ar g Leu Hi s Ty r Ser Gl y Ser Ile As n Al a Tr p Ser 1625 1630 1635 Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o 1640 1645 1650 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe 1655 1660 1665 Ser Ser Leu Ty r Ile Ser Gl n Phe Ile Ile Met Ty r Ser Leu As p 1670 1675 1680 Gl y Lys Lys Tr p Gl n Thr Ty r Ar g Gl y As n Ser Thr Gl y Thr Leu 1685 1690 1695 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n

   Page 268

   2018203206 08 May 2018

   1700 1705 97047 _1 1710 I l e Phe As n Pr o Pr o Ile Ile Al a Ar g Ty r Ile Ar g Leu Hi s Pr o 1715 1720 1725 Thr Hi s Ty r Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y 1730 1735 1740 Cy s As p Leu As n Ser Cy s Ser Met Pr o Leu Gl y Met Gl u Ser Lys 1745 1750 1755 Al a I l e Ser As p Al a Gl n Ile Thr Al a Ser Ser Ty r Phe Thr As n 1760 1765 1770 Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n 1775 1780 1785 Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u 1790 1795 1800 Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1805 1810 1815 Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Ty r Val Lys 1820 1825 1830

   Page 269

   97047 1

   2018203206 08 May 2018

   Gl u Phe 1835 Leu Ile Ser Ser Ser 1840 Gl n As p Gl y Hi s Gl n 1845 Tr p Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p 1850 1855 1860 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1865 1870 1875 Arg Tyr Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n I l e Al a 1880 1885 1890 Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p Leu Ty r As p 1895 1900 1905 Lys Thr Hi s Thr Cys Pr o Pr o Cy s Pr o Al a Pr o Gl u Leu Leu Gl y 1910 1915 1920 Gly Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1925 1930 1935 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cy s Val Val Val As p Val 1940 1945 1950 Ser Hi s Gl u Asp Pr o Gl u Val Lys Phe As n Tr p Ty r Val As p Gl y

   Page 270

   2018203206 08 May 2018

   1955 1960 97047 _1 1965 Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr 1970 1975 1980 As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n 1985 1990 1995 As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys 2000 2005 2010 Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a Lys Gl y 2015 2020 2025 Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p 2030 2035 2040 Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y 2045 2050 2055 Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n 2060 2065 2070 Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p 2075 2080 2085

   Page 271

   97047_1

   2018203206 08 May 2018

   Gl y Ser 2090 Phe Phe Leu Ty r Ser 2095 Lys Leu Thr Val As p 2100 Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser Cy s Ser Val Met Hi s Gl u Al a 2105 2110 2115 Leu Hi s As n Hi s Ty r Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y 2120 2125 2130

   Lys <210> 107 <211> 1984 <212> PRT <213> Artificial Sequence <220>

   <223> FVI I I 199 pr ot ei n sequence <400> 107

   Met Gl n Ile Gl u Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

   1 5 10 15

   Cys Phe Ser Al a Thr Arg Arg Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser

   20 25 30

   Page 272

   97047_1

   2018203206 08 May 2018

   Tr p As p Tyr 35 Met Gl n Ser As p Leu Gl y Gl u 40 Leu Pr o Val 45 As p Al a Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e 65 70 75 80 Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr I l e Gl n 85 90 95 Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser 100 105 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu 145 150 155 160 Lys Gl u Asn Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser

   Page 273

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   97047_1

   165 170 175

   Tyr Leu Ser Hi s 180 Val As p Leu Val Lys 185 As p Leu As n Ser Gl y 190 Leu I l e Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205 Gl n Thr Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y 210 215 220 Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p 225 230 235 240 Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr 245 250 255 Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val 260 265 270 Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285 Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser 290 295 300

   Page 274

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   97047 1

   Leu 305 Gl u I l e Ser Pr o I l e Thr 310 Phe Leu Thr Al a 315 Gl n Thr Leu Leu Met 320 Asp Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s 325 330 335 Asp Gly Met Gl u Al a Tyr Val Lys Val As p Ser Cy s Pr o Gl u Gl u Pr o 340 345 350 Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p 355 360 365 Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser 370 375 380 Pr o Ser Phe I l e Gl n Ile Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr 385 390 395 400 Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Ty r Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p As p Ar g Ser Ty r Lys Ser Gl n Ty r Leu As n 420 425 430 As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Ty r Lys Lys Val Ar g Phe Met

   Page 275

   2018203206 08 May 2018

   97047_1

   435 440 445

   Al a Tyr 450 Thr As p Gl u Thr Phe Lys Thr 455 Ar g Gl u Al a 460 I l e Gl n Hi s Gl u Ser Gl y I l e Leu Gl y Pr o Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu 465 470 475 480 Leu I l e I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n I l e Tyr Pr o 485 490 495 Hi s Gl y I l e Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys 500 505 510 Gl y Val Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe 515 520 525 Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p 530 535 540 Pr o Ar g Cys Leu Thr Ar g Tyr Tyr Ser Ser Phe Val As n Met Gl u Ar g 545 550 555 560 As p Leu Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cys Tyr Lys Gl u 565 570 575

   Page 276

   2018203206 08 May 2018

   97047_1

   Ser Val As p Gl n 580 Ar g Gl y As n Gl n Ile 585 Met Ser As p Lys Ar g 590 As n Val I l e Leu Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u 595 600 605 As n I l e Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p 610 615 620 Pr o Gl u Phe Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val 625 630 635 640 Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p 645 650 655 Ty r I l e Leu Ser Ile Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe 660 665 670 Ser Gl y Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr 675 680 685 Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o 690 695 700 Gl y Leu Tr p I l e Leu Gl y Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y

   Page 277

   2018203206 08 May 2018

   97047_1

   705 710 715 720

   Met Thr Al a Leu Leu 725 Lys Val Ser Ser Cy s 730 As p Lys As n Thr Gl y 735 As p Ty r Ty r Gl u As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys 740 745 750 Asn As n Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Pr o Pr o Val Leu 755 760 765 Lys Ar g Hi s Gl n Al a Gl u Ile Thr Ar g Thr Thr Leu Gl n Gl y Al a Pr o 770 775 780 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o 785 790 795 800 Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 805 810 815 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 820 825 830 Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Thr Pr o Gl y 835 840 845

   Page 278

   2018203206 08 May 2018

   97047_1

   Ser Gl y Thr 850 Al a Ser Ser Ser 855 Pr o Gl y Al a Ser Pr o 860 Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 865 870 875 880 Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Thr 885 890 895 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 900 905 910 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 915 920 925 Al a Ser Ser Ser As p Gl n Gl u Gl u Ile As p Ty r As p As p Thr I l e Ser 930 935 940 Val Gl u Met Lys Lys Gl u As p Phe As p Ile Ty r As p Gl u As p Gl u As n 945 950 955 960 Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Ty r Phe I l e Al a 965 970 975 Al a Val Gl u Ar g Leu Tr p As p Ty r Gl y Met Ser Ser Ser Pr o Hi s Val

   Page 279

   980

   2018203206 08 May 2018

   97047_1

   985 990

   Leu Arg Asn Arg Al a Gl n Ser Gl y Ser Val Pro Gl n Phe 1 Lys Lys Val 995 1000 1005 Val Phe Gl n Gl u Phe 1010 Thr Asp Gl y Ser Phe 1015 Thr Gl n Pr o 1020 Leu Ty r Ar g Gl y Gl u Leu Asn 1025 Gl u Hi s Leu Gl y Leu 1030 Leu Gl y Pr o 1035 Ty r I l e Ar g Al a Gl u Val Gl u 1040 Asp Asn Ile Met Val 1045 Thr Phe Ar g 1050 As n Gl n Al a Ser Arg Pro Tyr 1055 Ser Phe Tyr Ser Ser 1060 Leu I l e Ser 1065 Ty r Gl u Gl u As p Gl n Ar g Gl n 1070 Gl y Al a Gl u Pr o Ar g 1075 Lys Asn Phe 1080 Val Lys Pr o Asn Gl u Thr Lys 1085 Thr Tyr Phe Trp Lys 1090 Val Gl n Hi s 1095 Hi s Met Al a Pr o Thr Lys Asp 1100 Gl u Phe Asp Cys Lys 1105 Al a Tr p Al a 1110 Ty r Phe

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   97047_1

   Ser As p 1115 Val As p Leu Gl u Lys 1120 As p Val Hi s Ser Gl y 1125 Leu I l e Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y 1130 1135 1140 Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe 1145 1150 1155 As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u As n Met Gl u Ar g As n 1160 1165 1170 Cys Ar g Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 1175 1180 1185 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr 1190 1195 1200 Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 1205 1210 1215 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 1220 1225 1230 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a

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   97047 _1 1235 1240 1245 Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser 1250 1255 1260 Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 1265 1270 1275 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr 1280 1285 1290 Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 1295 1300 1305 Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Al a 1310 1315 1320 Ser Ser Al a Pr o Cys As n Ile Gl n Met Gl u As p Pr o Thr Phe Lys 1325 1330 1335 Gl u Asn Tyr Ar g Phe Hi s Al a Ile As n Gl y Tyr Ile Met As p Thr 1340 1345 1350 Leu Pr o Gl y Leu Val Met Al a Gl n Asp Gl n Ar g Ile Ar g Tr p Tyr 1355 1360 1365

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   97047 1

   Leu Leu 1370 Ser Met Gl y Ser As n 1375 Gl u As n Ile Hi s Ser 1380 I l e Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Tyr Lys Met 1385 1390 1395 Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe Gl u Thr Val Gl u Met 1400 1405 1410 Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys Leu I l e Gl y 1415 1420 1425 Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Tyr Ser 1430 1435 1440 Asn Lys Cys Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g 1445 1450 1455 As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Tyr Gl y Gl n Tr p Al a Pr o 1460 1465 1470 Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser Ile As n Al a Tr p Ser 1475 1480 1485 Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o

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   1490 1495 97047 _1 1500 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe 1505 1510 1515 Ser Ser Leu Tyr Ile Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p 1520 1525 1530 Gly Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu 1535 1540 1545 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n 1550 1555 1560 I l e Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr Ile Ar g Leu Hi s Pr o 1565 1570 1575 Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y 1580 1585 1590 Cys As p Leu As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys 1595 1600 1605 Al a I l e Ser As p Al a Gl n Ile Thr Al a Ser Ser Tyr Phe Thr As n 1610 1615 1620

   Page 284

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   Met Phe 1625 Al a Thr Tr p Ser Pr o 1630 Ser Lys Al a Ar g Leu 1635 Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u 1640 1645 1650 Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1655 1660 1665 Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Ty r Val Lys 1670 1675 1680 Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu 1685 1690 1695 Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p 1700 1705 1710 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1715 1720 1725 Ar g Ty r Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n I l e Al a 1730 1735 1740 Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p Leu Ty r As p

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   1745 1750 97047 _1 1755 Lys Thr Hi s Thr Cys Pr o Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y 1760 1765 1770 Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1775 1780 1785 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val 1790 1795 1800 Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Tyr Val As p Gl y 1805 1810 1815 Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr 1820 1825 1830 As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n 1835 1840 1845 As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys 1850 1855 1860 Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a Lys Gl y 1865 1870 1875

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   Gl n Pr o 1880 Ar g Gl u Pr o Gl n Val 1885 Tyr Thr Leu Pr o Pr o 1890 Ser Ar g As p Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y 1895 1900 1905 Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n 1910 1915 1920 Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p 1925 1930 1935 Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g 1940 1945 1950 Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a 1955 1960 1965 Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y 1970 1975 1980

   Lys <210> 108

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   2018203206 08 May 2018

   97047_1 <211> 2134 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> FVI I I 201 pr ot ei n sequence <400> 108

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cys Phe Phe 10 Leu Cys Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val Gl y Al a Pr o 35 40 45 Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o 50 55 60 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 65 70 75 80 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 85 90 95 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o

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   100 105 97047_1 110 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 115 120 125 Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o 130 135 140 Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Ser Ser Thr 145 150 155 160 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 165 170 175 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 180 185 190 Al a Ser Ser As p Al a Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o 195 200 205 Phe As n Thr Ser Val Val Ty r Lys Lys Thr Leu Phe Val Gl u Phe Thr 210 215 220 As p Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu 225 230 235 240

   Page 289

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   Leu Gl y Pr o Thr Ile 245 Gl n Al a Gl u Val Ty r 250 As p Thr Val Val I l e 255 Thr Leu Lys As n Met Al a Ser Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val 260 265 270 Ser Ty r Tr p Lys Al a Ser Gl u Gl y Al a Gl u Ty r As p As p Gl n Thr Ser 275 280 285 Gl n Ar g Gl u Lys Gl u As p As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr 290 295 300 Ty r Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o 305 310 315 320 Leu Cy s Leu Thr Ty r Ser Ty r Leu Ser Hi s Val As p Leu Val Lys As p 325 330 335 Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cy s Ar g Gl u Gl y Ser 340 345 350 Leu Al a Lys Gl u Lys Thr Gl n Thr Leu Hi s Lys Phe I l e Leu Leu Phe 355 360 365 Al a Val Phe As p Gl u Gl y Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser

   Page 290

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   97047_1

   370 375 380

   Leu 385 Met Gl n As p Ar g As p Al a Al a 390 Ser Al a Ar g 395 Al a Tr p Pr o Lys Met 400 Hi s Thr Val As n Gl y Ty r Val As n Ar g Ser Leu Pr o Gl y Leu I l e Gl y 405 410 415 Cy s Hi s Ar g Lys Ser Val Ty r Tr p Hi s Val Ile Gl y Met Gl y Thr Thr 420 425 430 Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g 435 440 445 Asn Hi s Ar g Gl n Al a Ser Leu Gl u Ile Ser Pr o Ile Thr Phe Leu Thr 450 455 460 Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu Leu Phe Cy s Hi s 465 470 475 480 I l e Ser Ser Hi s Gl n Hi s As p Gl y Met Gl u Al a Ty r Val Lys Val As p 485 490 495 Ser Cy s Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a 500 505 510

   Page 291

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   Gl u As p Tyr 515 As p As p As p Leu Thr 520 As p Ser Gl u Met As p 525 Val Val Ar g Phe As p As p As p As n Ser Pr o Ser Phe Ile Gl n Ile Ar g Ser Val Al a 530 535 540 Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u 545 550 555 560 As p Tr p As p Tyr Al a Pr o Leu Val Leu Al a Pr o As p As p Ar g Ser Tyr 565 570 575 Lys Ser Gl n Tyr Leu As n As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr 580 585 590 Lys Lys Val Ar g Phe Met Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g 595 600 605 Gl u Al a I l e Gl n Hi s Gl u Ser Gl y Ile Leu Gl y Pr o Leu Leu Tyr Gl y 610 615 620 Gl u Val Gl y As p Thr Leu Leu Ile Ile Phe Lys As n Gl n Al a Ser Ar g 625 630 635 640 Pr o Tyr As n I l e Tyr Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Tyr

   Page 292

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   97047_1

   645 650 655

   Ser Ar g Ar g Leu 660 Pr o Lys Gl y Val Lys 665 Hi s Leu Lys As p Phe 670 Pr o I l e Leu Pr o Gl y Gl u Ile Phe Lys Ty r Lys Tr p Thr Val Thr Val Gl u As p 675 680 685 Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s Leu Thr Ar g Ty r Ty r Ser Ser 690 695 700 Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu 705 710 715 720 Leu I l e Cy s Ty r Lys Gl u Ser Val As p Gl n Ar g Gl y As n Gl n I l e Met 725 730 735 Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe As p Gl u As n Ar g 740 745 750 Ser Tr p Ty r Leu Thr Gl u As n Ile Gl n Ar g Phe Leu Pr o As n Pr o Al a 755 760 765 Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser As n I l e Met Hi s 770 775 780

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   Ser 785 I l e As n Gl y Tyr Val 790 Phe As p Ser Leu Gl n 795 Leu Ser Val Cys Leu 800 Hi s Gl u Val Al a Tyr Tr p Tyr Ile Leu Ser Ile Gl y Al a Gl n Thr As p 805 810 815 Phe Leu Ser Val Phe Phe Ser Gl y Tyr Thr Phe Lys Hi s Lys Met Val 820 825 830 Tyr Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe 835 840 845 Met Ser Met Gl u As n Pr o Gl y Leu Tr p Ile Leu Gl y Cys Hi s As n Ser 850 855 860 As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cys 865 870 875 880 As p Lys As n Thr Gl y As p Tyr Tyr Gl u As p Ser Tyr Gl u As p I l e Ser 885 890 895 Al a Tyr Leu Leu Ser Lys As n As n Al a Ile Gl u Pr o Ar g Ser Phe Ser 900 905 910 Gl n As n Pr o Pr o Val Leu Lys Ar g Hi s Gl n Al a Gl u I l e Thr Ar g Thr

   Page 294

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   915 Gl y Al a Pr o 920 Gl y Ser 925 Ser Ser Thr Leu 930 Gl n Gl y Thr 935 Pr o Gl y Thr 940 Al a Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a 945 950 955 960 Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr 965 970 975 Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr 980 985 990 Gl y Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a 995 1000 1005 Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl ^y Al a Ser Pr o Gl y 1010 1015 1020 Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 1025 1030 1035 Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl ^y Al a Thr Gl y Ser 1040 1045 1050

   Page 295

   97047_1

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   Pr o Gl y Ser Ser Thr Pr o Ser 1060 Gl y Al a Thr Gl y Ser 1065 Pr o Gl y Al a 1055 Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Al a Ser Ser Ser As p 1070 1075 1080 Gl n Gl u Gl u Ile As p Ty r As p As p Thr Ile Ser Val Gl u Met Lys 1085 1090 1095 Lys Gl u As p Phe As p Ile Ty r As p Gl u As p Gl u As n Gl n Ser Pr o 1100 1105 1110 Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Ty r Phe Ile Al a Al a Val 1115 1120 1125 Gl u Ar g Leu Tr p As p Ty r Gl y Met Ser Ser Ser Pr o Hi s Val Leu 1130 1135 1140 Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o Gl n Phe Lys Lys Val 1145 1150 1155 Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe Thr Gl n Pr o Leu Ty r 1160 1165 1170 Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu Leu Gl y Pr o Ty r I l e

   Page 296

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   97047_1

   1175 1180 1185

   Arg Al a Gl u Val Gl u As p As n 1195 Ile Met Val Thr Phe 1200 Ar g As n Gl n 1190 Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Gl u 1205 1210 1215 Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g Lys As n Phe Val Lys 1220 1225 1230 Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met 1235 1240 1245 Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys Al a Tr p Al a Tyr Phe 1250 1255 1260 Ser As p Val Asp Leu Gl u Lys As p Val Hi s Ser Gl y Leu I l e Gl y 1265 1270 1275 Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y 1280 1285 1290 Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe

   1295 1300 1305

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   As p Gl u Thr Lys Ser Tr p Tyr 1315 Phe Thr Gl u As n Met 1320 Gl u Ar g As n 1310 Cys Ar g Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 1325 1330 1335 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr 1340 1345 1350 Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 1355 1360 1365 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 1370 1375 1380 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a 1385 1390 1395 Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser 1400 1405 1410 Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 1415 1420 1425 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr

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   Ser

   Pr o

   Ser

   Gl u

   Leu

   Leu

   Ser

   Al a

   1430

   1435

   1440

   Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 1445 1450 1455

   Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Al a 1460 1465 1470

   Ser Al a Pro Cys Asn Ile Gl n Met Gl u Asp Pro Thr Phe Lys 1475 1480 1485

   Asn Tyr Arg Phe Hi s Al a Ile Asn Gl y Tyr I l e Met Asp Thr 1490 1495 1500

   Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr 1505 1510 1515

   Leu Ser Met Gl y Ser As n Gl u As n I l e Hi s Ser I l e Hi s Phe 1520 1525 1530

   Gl y Hi s Val Phe Thr Val Arg Lys Lys Gl u Gl u Tyr Lys Met 1535 1540 1545

   Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met

   1550 1555 1560

   Page 299

   97047 1

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   Leu Pr o 1565 Ser Lys Al a Gl y Ile 1570 Tr p Ar g Val Gl u Cys 1575 Leu I l e Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Tyr Ser 1580 1585 1590 As n Lys Cys Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g 1595 1600 1605 As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Tyr Gl y Gl n Tr p Al a Pr o 1610 1615 1620 Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser Ile As n Al a Tr p Ser 1625 1630 1635 Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o 1640 1645 1650 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe 1655 1660 1665 Ser Ser Leu Tyr Ile Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p 1670 1675 1680 Gl y Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu

   Page 300

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   1685 1690 97047 _1 1695 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n 1700 1705 1710 I l e Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr Ile Ar g Leu Hi s Pr o 1715 1720 1725 Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y 1730 1735 1740 Cys As p Leu As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys 1745 1750 1755 Al a I l e Ser As p Al a Gl n Ile Thr Al a Ser Ser Tyr Phe Thr As n 1760 1765 1770 Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n 1775 1780 1785 Gly Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u 1790 1795 1800 Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1805 1810 1815

   Page 301

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   Thr Thr 1820 Gl n Gl y Val Lys Ser 1825 Leu Leu Thr Ser Met 1830 Tyr Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n Asp Gl y Hi s Gl n Tr p Thr Leu 1835 1840 1845 Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p 1850 1855 1860 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1865 1870 1875 Ar g Tyr Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n I l e Al a 1880 1885 1890 Leu Ar g Met Gl u Val Leu Gl y Cys Gl u Al a Gl n As p Leu Tyr As p 1895 1900 1905 Lys Thr Hi s Thr Cys Pr o Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y 1910 1915 1920 Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1925 1930 1935 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val

   Page 302

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   97047_1

   1940 1945 1950

   Ser Hi s 1955 Gl u As p Pr o Gl u Val 1960 Lys Phe As n Tr p Tyr 1965 Val As p Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr 1970 1975 1980 As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n 1985 1990 1995 As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys 2000 2005 2010 Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a Lys Gl y 2015 2020 2025 Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p 2030 2035 2040 Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y 2045 2050 2055 Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n

   2060 2065 2070

   Page 303

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   Pr o Gl u 2075 As n As n Tyr Lys Thr 2080 Thr Pr o Pr o Val Leu 2085 As p Ser As p Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g 2090 2095 2100 Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a 2105 2110 2115 Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y 2120 2125 2130

   Lys

   <210> 109 <211> 2128 <212> PRT <213> Ar t i f i ci al Sequence <220> <223> FVI I I 203 pr ot ei n sequence

   <400> 109

   Met Gl n I l e Gl u Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

   1 5 10 15

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   97047_1

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   Cys Phe Ser Al a 20 Thr Arg Arg Tyr Tyr 25 Leu Gl y Al a Val Gl u 30 Leu Ser Tr p As p Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g 35 40 45 Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e 65 70 75 80 Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr I l e Gl n 85 90 95 Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser 100 105 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu

   Page 305

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   97047_1

   145 150 155 160

   Lys Gl u As n Gl y Pr o 165 Met Al a Ser As p Pr o Leu 170 Cy s Leu Thr Ty r 175 Ser Ty r Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e 180 185 190 Gl y Al a Leu Leu Val Cy s Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205 Gl n Thr Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y 210 215 220 Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p 225 230 235 240 Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Ty r 245 250 255 Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cy s Hi s Ar g Lys Ser Val 260 265 270 Ty r Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285

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   Phe Leu Gl u Gl y Hi s Thr Phe 295 Leu Val Ar g As n Hi s 300 Ar g Gl n Al a Ser 290 Leu Gl u I l e Ser Pr o Ile Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met 305 310 315 320 As p Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s 325 330 335 As p Gl y Met Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o 340 345 350 Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Tyr As p As p As p 355 360 365 Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser 370 375 380 Pr o Ser Phe I l e Gl n Ile Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr 385 390 395 400 Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n

   Page 307

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   970 47_1 420 425 430 As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Ty r Lys Lys Val Ar g Phe Met 435 440 445 Al a Ty r Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a I l e Gl n Hi s Gl u 450 455 460 Ser Gl y I l e Leu Gl y Pr o Leu Leu Ty r Gl y Gl u Val Gl y As p Thr Leu 465 470 475 480 Leu I l e I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Ty r As n I l e Ty r Pr o 485 490 495 Hi s Gl y I l e Thr As p Val Ar g Pr o Leu Ty r Ser Ar g Ar g Leu Pr o Lys 500 505 510 Gl y Val Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe 515 520 525 Lys Ty r Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p 530 535 540 Pr o Ar g Cy s Leu Thr Ar g Ty r Ty r Ser Ser Phe Val As n Met Gl u Ar g 545 550 555 560

   Page 308

   97047_1

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   As p Leu Al a Ser Gl y 565 Leu Ile Gl y Pr o Leu 570 Leu Ile Cy s Ty r Lys 575 Gl u Ser Val As p Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val 580 585 590 I l e Leu Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u 595 600 605 As n I l e Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p 610 615 620 Pr o Gl u Phe Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val 625 630 635 640 Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p 645 650 655 Ty r I l e Leu Ser Ile Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe 660 665 670 Ser Gl y Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr 675 680 685 Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o

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   97047_1

   690 695 700

   Gl y 705 Leu Tr p I l e Leu Gl y 710 Cy s Hi s As n Ser As p 715 Phe Ar g As n Ar g Gl y 720 Met Thr Al a Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p 725 730 735 Ty r Ty r Gl u As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys 740 745 750 As n As n Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Gl y Al a Pr o Gl y 755 760 765 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 770 775 780 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 785 790 795 800 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 805 810 815 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u 820 825 830

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   Pr o

   Thr

   Ser

   865

   Al a

   Thr

   Thr

   Al a

   Ser

   945

   97047_1

   Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 835 840 845

   Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 850 855 860

   Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser 870 875 880

   Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 885 890 895

   Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y 900 905 910

   Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser 915 920 925

   Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 930 935 940

   Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 950 955 960

   Ser

   Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a

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   965 Thr 970 Pr o Gl y Ser Pr o Al a Gl y Ser 985 975 Thr Ser Gl y Ser 980 Gl u Pr o Thr 990 Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 995 1000 1005 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o 1010 1015 1020 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr 1025 1030 1035 Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser 1040 1045 1050 Al a Pr o Al a Ser Ser Pr o Pr o Val Leu Lys Ar g Hi s Gl n Al a Gl u 1055 1060 1065 I l e Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u I l e As p Ty r 1070 1075 1080 As p As p Thr Ile Ser Val Gl u Met Ly s Ly s Gl u As p Phe As p I l e 1085 1090 1095

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   Tyr As p 1100 Gl u As p Gl u As n Gl n 1105 Ser Pr o Ar g Ser Phe 1110 Gl n Lys Lys Thr Ar g Hi s Tyr Phe Ile Al a Al a Val Gl u Ar g Leu Tr p As p Tyr 1115 1120 1125 Gly Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser 1130 1135 1140 Gly Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr 1145 1150 1155 Asp Gl y Ser Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u 1160 1165 1170 Hi s Leu Gl y Leu Leu Gl y Pr o Tyr Ile Ar g Al a Gl u Val Gl u As p 1175 1180 1185 As n I l e Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser 1190 1195 1200 Phe Tyr Ser Ser Leu Ile Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y 1205 1210 1215 Al a Gl u Pr o Ar g Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr

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   1220 1225 97047 _1 1230 Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u 1235 1240 1245 Phe As p Cys Lys Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u 1250 1255 1260 Lys As p Val Hi s Ser Gl y Leu Ile Gl y Pr o Leu Leu Val Cys Hi s 1265 1270 1275 Thr As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n 1280 1285 1290 Gl u Phe Al a Leu Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p 1295 1300 1305 Tyr Phe Thr Gl u As n Met Gl u Ar g As n Cys Ar g Gl y Al a Pr o Thr 1310 1315 1320 Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 1325 1330 1335 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 1340 1345 1350

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   Gl u Ser 1355 Gl y Pr o Gl y Ser Gl u 1360 Pr o Al a Thr Ser Gl y 1365 Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr 1370 1375 1380 Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser 1385 1390 1395 Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 1400 1405 1410 Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u 1415 1420 1425 Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr 1430 1435 1440 Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u 1445 1450 1455 Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Al a Ser Ser Al a Pr o Cy s As n 1460 1465 1470 I l e Gl n Met Gl u As p Pr o Thr Phe Lys Gl u As n Ty r Ar g Phe Hi s

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   1475 1480 97047 _1 1485 Al a I l e As n Gl y Tyr Ile Met As p Thr Leu Pr o Gl y Leu Val Met 1490 1495 1500 Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr Leu Leu Ser Met Gl y Ser 1505 1510 1515 As n Gl u As n Ile Hi s Ser Ile Hi s Phe Ser Gl y Hi s Val Phe Thr 1520 1525 1530 Val Ar g Lys Lys Gl u Gl u Tyr Lys Met Al a Leu Tyr As n Leu Tyr 1535 1540 1545 Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y 1550 1555 1560 I l e Tr p Ar g Val Gl u Cys Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y 1565 1570 1575 Met Ser Thr Leu Phe Leu Val Tyr Ser As n Lys Cys Gl n Thr Pr o 1580 1585 1590 Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n I l e Thr Al a 1595 1600 1605

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   Ser Gl y 1610 Gl n Tyr Gl y Gl n Tr p 1615 Al a Pr o Lys Leu Al a 1620 Ar g Leu Hi s Tyr Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser 1625 1630 1635 Tr p I l e Lys Val As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y I l e 1640 1645 1650 Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu Tyr I l e Ser 1655 1660 1665 Gl n Phe I l e Ile Met Tyr Ser Leu As p Gl y Lys Lys Tr p Gl n Thr 1670 1675 1680 Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n 1685 1690 1695 Val As p Ser Ser Gl y Ile Lys Hi s As n Ile Phe As n Pr o Pr o I l e 1700 1705 1710 I l e Al a Ar g Tyr Ile Ar g Leu Hi s Pr o Thr Hi s Tyr Ser I l e Ar g 1715 1720 1725 Ser Thr Leu Ar g Met Gl u Leu Met Gl y Cys As p Leu As n Ser Cys

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   9 7047 _1 1730 1735 1740 Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n 1745 1750 1755 I l e Thr Al a Ser Ser Tyr Phe Thr As n Met Phe Al a Thr Tr p Ser 1760 1765 1770 Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p 1775 1780 1785 Ar g Pr o Gl n Val As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe 1790 1795 1800 Gl n Lys Thr Met Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys 1805 1810 1815 Ser Leu Leu Thr Ser Met Tyr Val Lys Gl u Phe Leu I l e Ser Ser 1820 1825 1830 Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys 1835 1840 1845 Val Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val 1850 1855 1860

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   As n Ser 1865 Leu As p Pr o Pr o Leu 1870 Leu Thr Ar g Tyr Leu 1875 Ar g I l e Hi s Pr o Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu 1880 1885 1890 Gl y Cys Gl u Al a Gl n As p Leu Tyr As p Lys Thr Hi s Thr Cys Pr o 1895 1900 1905 Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu 1910 1915 1920 Phe Pr o Pr o Lys Pr o Lys As p Thr Leu Met Ile Ser Ar g Thr Pr o 1925 1930 1935 Gl u Val Thr Cys Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u 1940 1945 1950 Val Lys Phe As n Tr p Tyr Val As p Gl y Val Gl u Val Hi s As n Al a 1955 1960 1965 Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr As n Ser Thr Tyr Ar g Val 1970 1975 1980 Val Ser Val Leu Thr Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys

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   1985 1990 97047 _1 1995 Gl u Tyr Lys Cys Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o I l e 2000 2005 2010 Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n 2015 2020 2025 Val Tyr Thr Leu Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n 2030 2035 2040 Val Ser Leu Thr Cys Leu Val Lys Gl y Phe Tyr Pr o Ser As p I l e 2045 2050 2055 Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Tyr Lys 2060 2065 2070 Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe Phe Leu Tyr 2075 2080 2085 Ser Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val 2090 2095 2100 Phe Ser Cys Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr 2105 2110 2115

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   97047_1

   Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y Lys 2120 2125 <210> 110 <211> 2128 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> FVI I I 204 pr ot ei n sequence <400> 110

   Met 1 Gl n I l e Gl u Leu Ser 5 Thr Cys Phe Phe 10 Leu Cys Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g 35 40 45 Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val 50 55 60 Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e 65 70 75 80

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   Al a Lys Pr o Ar g Pr o Pr o Tr p 85 Met Gl y Leu 90 Leu Gl y Pr o Thr I l e 95 Gl n Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser 100 105 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu 145 150 155 160 Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e 180 185 190 Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205 Gl n Thr Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y

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   210 215 220

   Lys 225 Ser Tr p Hi s Ser Gl u 230 Thr Lys As n Ser Leu 235 Met Gl n As p Ar g As p 240 Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr 245 250 255 Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val 260 265 270 Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285 Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser 290 295 300 Leu Gl u I l e Ser Pr o Ile Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met 305 310 315 320 As p Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s 325 330 335 As p Gl y Met Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o 340 345 350

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   Gl n Leu Ar g 355 Met Lys As n As n Gl u 360 Gl u Al a Gl u Asp Tyr 365 As p As p As p Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser 370 375 380 Pr o Ser Phe I l e Gl n Ile Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr 385 390 395 400 Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p Gl y Al a Pr o Thr Ser Thr Gl u Pr o Ser Gl u 420 425 430 Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 435 440 445 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 450 455 460 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 465 470 475 480 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o

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   97047_1

   485 490 495

   Gly Thr Ser Gl u 500 Ser Al a Thr Pr o Gl u 505 Ser Gl y Pr o Gl y Ser 510 Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u 515 520 525 Gl y Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 530 535 540 Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u 545 550 555 560 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Al a Ser Ser As p Ar g Ser Tyr 565 570 575 Lys Ser Gl n Tyr Leu As n As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr 580 585 590 Lys Lys Val Ar g Phe Met Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g 595 600 605 Gl u Al a I l e Gl n Hi s Gl u Ser Gl y Ile Leu Gl y Pr o Leu Leu Tyr Gl y 610 615 620

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   Gl u Val 625 Gl y As p Thr Leu 630 Leu Ile I l e Phe Lys Asn Gl n 635 Al a Ser Ar g 640 Pr o Tyr As n I l e Tyr Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Tyr 645 650 655 Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o I l e 660 665 670 Leu Pr o Gl y Gl u Ile Phe Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p 675 680 685 Gl y Pr o Thr Lys Ser As p Pr o Ar g Cys Leu Thr Ar g Tyr Tyr Ser Ser 690 695 700 Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu 705 710 715 720 Leu I l e Cys Tyr Lys Gl u Ser Val As p Gl n Ar g Gl y As n Gl n I l e Met 725 730 735 Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe As p Gl u As n Ar g 740 745 750 Ser Tr p Tyr Leu Thr Gl u As n Ile Gl n Ar g Phe Leu Pr o As n Pr o Al a

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   97047_1

   755 760 765

   Gl y Val 770 Gl n Leu Gl u As p Pr o 775 Gl u Phe Gl n Al a Ser 780 As n I l e Met Hi s Ser I l e As n Gl y Ty r Val Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu 785 790 795 800 Hi s Gl u Val Al a Ty r Tr p Ty r Ile Leu Ser Ile Gl y Al a Gl n Thr As p 805 810 815 Phe Leu Ser Val Phe Phe Ser Gl y Ty r Thr Phe Lys Hi s Lys Met Val 820 825 830 Ty r Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe 835 840 845 Met Ser Met Gl u As n Pr o Gl y Leu Tr p Ile Leu Gl y Cy s Hi s As n Ser 850 855 860 As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cy s 865 870 875 880 As p Lys As n Thr Gl y As p Ty r Ty r Gl u As p Ser Ty r Gl u As p I l e Ser 885 890 895

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   Al a Tyr Leu Leu Ser Ly s As n As n Al a Ile Gl u Pr o Ar g Se r Phe Ser 900 905 91 0 Gl n As n Gl y Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 915 920 925 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl ^y Thr Ser 930 935 940 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser 945 95 0 955 960 Gl y Ser Gl u Thr Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 965 970 975 Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Ser Pr o 980 985 99 0 Al a Gl y Ser Pr o Thr Se r Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr 995 1000 10! 05

   Pro Gl u Ser Gl y Pro Gl y Ser Gl u 1010 1015

   Pr o Al a Thr

   Ser Gl y Ser Gl u 1020

   Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser

   Gl y Pr o Gl y

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   97047_1

   1025 1030 1035

   Ser Pr o 1040 Al a Gl y Ser Pr o Thr 1045 Ser Thr Gl u Gl u Gl y 1050 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser 1055 1060 1065 Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser 1070 1075 1080 Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 1085 1090 1095 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 1100 1105 1110 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser 1115 1120 1125 Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr 1130 1135 1140 Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y

   1145 1150 1155

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   Thr Ser 1160 Thr Gl u Pr o Ser Gl u 1165 Gl y Ser Al a Pr o Gl y 1170 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr 1175 1180 1185 Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser 1190 1195 1200 Al a Pr o Al a Ser Ser Pr o Pr o Val Leu Lys Ar g Hi s Gl n Al a Gl u 1205 1210 1215 I l e Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u I l e As p Tyr 1220 1225 1230 As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u As p Phe As p I l e 1235 1240 1245 Tyr As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys 1250 1255 1260 Thr Ar g Hi s Tyr Phe Ile Al a Al a Val Gl u Ar g Leu Tr p As p Tyr 1265 1270 1275 Gl y Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser

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   1280 1285 97047 _1 1290 Gl y Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr 1295 1300 1305 As p Gl y Ser Phe Thr Gl n Pr o Leu Ty r Ar g Gl y Gl u Leu As n Gl u 1310 1315 1320 Hi s Leu Gl y Leu Leu Gl y Pr o Ty r Ile Ar g Al a Gl u Val Gl u As p 1325 1330 1335 As n I l e Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Ty r Ser 1340 1345 1350 Phe Ty r Ser Ser Leu Ile Ser Ty r Gl u Gl u As p Gl n Ar g Gl n Gl y 1355 1360 1365 Al a Gl u Pr o Ar g Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr 1370 1375 1380 Ty r Phe Tr p Lys Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u 1385 1390 1395 Phe As p Cy s Lys Al a Tr p Al a Ty r Phe Ser As p Val As p Leu Gl u 1400 1405 1410

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   Lys As p 1415 Val Hi s Ser Gl y Leu 1420 Ile Gl y Pr o Leu Leu 1425 Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n 1430 1435 1440 Gl u Phe Al a Leu Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p 1445 1450 1455 Tyr Phe Thr Gl u As n Met Gl u Ar g As n Cys Ar g Al a Pr o Cys As n 1460 1465 1470 I l e Gl n Met Gl u As p Pr o Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s 1475 1480 1485 Al a I l e As n Gl y Tyr Ile Met As p Thr Leu Pr o Gl y Leu Val Met 1490 1495 1500 Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr Leu Leu Ser Met Gl y Ser 1505 1510 1515 As n Gl u As n Ile Hi s Ser Ile Hi s Phe Ser Gl y Hi s Val Phe Thr 1520 1525 1530 Val Ar g Lys Lys Gl u Gl u Tyr Lys Met Al a Leu Tyr As n Leu Tyr

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   1535 1540 97047 _1 1545 Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y 1550 1555 1560 I l e Tr p Ar g Val Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y 1565 1570 1575 Met Ser Thr Leu Phe Leu Val Ty r Ser As n Lys Cy s Gl n Thr Pr o 1580 1585 1590 Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n I l e Thr Al a 1595 1600 1605 Ser Gl y Gl n Ty r Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s 1610 1615 1620 Ty r Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser 1625 1630 1635 Tr p I l e Lys Val As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y I l e 1640 1645 1650 Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r I l e Ser 1655 1660 1665

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   Gl n Phe 1670 I l e Ile Met Tyr Ser 1675 Leu As p Gl y Lys Lys 1680 Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n 1685 1690 1695 Val As p Ser Ser Gl y Ile Lys Hi s As n Ile Phe As n Pr o Pr o I l e 1700 1705 1710 I l e Al a Ar g Tyr Ile Ar g Leu Hi s Pr o Thr Hi s Tyr Ser I l e Ar g 1715 1720 1725 Ser Thr Leu Ar g Met Gl u Leu Met Gl y Cys As p Leu As n Ser Cys 1730 1735 1740 Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n 1745 1750 1755 I l e Thr Al a Ser Ser Tyr Phe Thr As n Met Phe Al a Thr Tr p Ser 1760 1765 1770 Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p 1775 1780 1785 Ar g Pr o Gl n Val As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe

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   1790 1795 97047 _1 1800 Gl n Lys Thr Met Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys 1805 1810 1815 Ser Leu Leu Thr Ser Met Ty r Val Lys Gl u Phe Leu I l e Ser Ser 1820 1825 1830 Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys 1835 1840 1845 Val Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val 1850 1855 1860 As n Ser Leu As p Pr o Pr o Leu Leu Thr Ar g Ty r Leu Ar g I l e Hi s 1865 1870 1875 Pr o Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu 1880 1885 1890 Gl y Cys Gl u Al a Gl n As p Leu Ty r As p Lys Thr Hi s Thr Cy s Pr o 1895 1900 1905 Pr o Cy s Pr o Al a Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu 1910 1915 1920

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   Phe Pr o 1925 Pr o Lys Pr o Ly s As p 1930 Thr Leu Met Ile Ser 1935 Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u 1940 1945 1950 Val Lys Phe As n Tr p Tyr Val As p Gl y Val Gl u Val Hi s As n Al a 1955 1960 1965 Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr As n Ser Thr Tyr Ar g Val 1970 1975 1980 Val Ser Val Leu Thr Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys 1985 1990 1995 Gl u Tyr Lys Cys Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o I l e 2000 2005 2010 Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n 2015 2020 2025 Val Tyr Thr Leu Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n 2030 2035 2040 Val Ser Leu Thr Cys Leu Val Lys Gl y Phe Tyr Pr o Ser As p I l e

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   2045 2050 2055 Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Tyr Lys 2060 2065 2070 Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe Phe Leu Tyr 2075 2080 2085 Ser Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val 2090 2095 2100 Phe Ser Cys Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr 2105 2110 2115

   Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y Lys 2120 2125 <210> 111 <211> 2128 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> FVI I I 205 pr ot ei n sequence <400> 111

   Met Gl n Ile Gl u Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

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   97047_1

   1 5 10 15

   Cys Phe Ser Al a 20 Thr Ar g Ar g Tyr Tyr 25 Leu Gl y Al a Val Gl u 30 Leu Ser Tr p As p Tyr Met Gl n Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u 35 40 45 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 50 55 60 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 65 70 75 80 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 85 90 95 Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 100 105 110 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser 115 120 125 Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser 130 135 140

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   Gl u 145 Thr Pr o Gl y Thr Ser 150 Gl u Ser Al a Thr Pr o 155 Gl u Ser Gl y Pr o Gl y 160 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 165 170 175 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Al a Ser Ser Ser As p Leu Gl y Gl u 180 185 190 Leu Pr o Val As p Al a Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o 195 200 205 Phe Asn Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr 210 215 220 Asp Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu 225 230 235 240 Leu Gl y Pr o Thr Ile Gl n Al a Gl u Val Tyr As p Thr Val Val I l e Thr 245 250 255 Leu Lys As n Met Al a Ser Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val 260 265 270 Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser

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   97047_1

   275 280 285

   Gl n Ar g Gl u Lys Gl u As p As p 295 Lys Val Phe Pr o Gl y Gl y Ser 300 Hi s Thr 290 Tyr Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o 305 310 315 320 Leu Cys Leu Thr Tyr Ser Tyr Leu Ser Hi s Val As p Leu Val Lys As p 325 330 335 Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser 340 345 350 Leu Al a Lys Gl u Lys Thr Gl n Thr Leu Hi s Lys Phe I l e Leu Leu Phe 355 360 365 Al a Val Phe As p Gl u Gl y Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser 370 375 380 Leu Met Gl n As p Ar g As p Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met 385 390 395 400 Hi s Thr Val As n Gl y Tyr Val As n Ar g Ser Leu Pr o Gl y Leu I l e Gl y 405 410 415

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   Cys Hi s Ar g Lys 420 Ser Val Tyr Tr p Hi s 425 Val Ile Gl y Met Gl y 430 Thr Thr Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g 435 440 445 As n Hi s Ar g Gl n Al a Ser Leu Gl u Ile Ser Pr o Ile Thr Phe Leu Thr 450 455 460 Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s 465 470 475 480 I l e Ser Ser Hi s Gl n Hi s As p Gl y Met Gl u Al a Tyr Val Lys Val As p 485 490 495 Ser Cys Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a 500 505 510 Gl u As p Tyr As p As p As p Leu Thr As p Ser Gl u Met As p Val Val Ar g 515 520 525 Phe As p As p As p As n Ser Pr o Ser Phe Ile Gl n Ile Ar g Ser Val Al a 530 535 540 Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u

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   97047_1

   545 550 555 560

   As p Tr p As p Tyr Al a 565 Pr o Leu Val Leu Al a Pr o Asp Asp Ar g Ser Tyr 570 575 Lys Ser Gl n Tyr Leu As n As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr 580 585 590 Lys Lys Val Ar g Phe Met Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g 595 600 605 Gl u Al a I l e Gl n Hi s Gl u Ser Gl y Ile Leu Gl y Pr o Leu Leu Tyr Gl y 610 615 620 Gl u Val Gl y As p Thr Leu Leu Ile Ile Phe Lys As n Gl n Al a Ser Ar g 625 630 635 640 Pr o Tyr As n I l e Tyr Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Tyr 645 650 655 Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o I l e 660 665 670 Leu Pr o Gl y Gl u Ile Phe Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p 675 680 685

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   Gl y Pr o Thr 690 Lys Ser As p Pr o 695 Ar g Cys Leu Thr Ar g 700 Tyr Tyr Ser Ser Phe Val Asn Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu 705 710 715 720 Leu I l e Cys Tyr Lys Gl u Ser Val As p Gl n Ar g Gl y As n Gl n I l e Met 725 730 735 Ser As p Lys Arg As n Val Ile Leu Phe Ser Val Phe As p Gl u As n Ar g 740 745 750 Ser Tr p Tyr Leu Thr Gl u As n Ile Gl n Ar g Phe Leu Pr o As n Pr o Al a 755 760 765 Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser As n I l e Met Hi s 770 775 780 Ser I l e As n Gl y Tyr Val Phe As p Ser Leu Gl n Leu Ser Val Cys Leu 785 790 795 800 Hi s Gl u Val Al a Tyr Tr p Tyr Ile Leu Ser Ile Gl y Al a Gl n Thr As p 805 810 815 Phe Leu Ser Val Phe Phe Ser Gl y Tyr Thr Phe Lys Hi s Lys Met Val

   Page 343

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   820 825 97047_1 830 Ty r Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe 835 840 845 Met Ser Met Gl u As n Pr o Gl y Leu Tr p Ile Leu Gl y Cy s Hi s As n Ser 850 855 860 As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cy s 865 870 875 880 As p Lys As n Thr Gl y As p Ty r Ty r Gl u As p Ser Ty r Gl u As p I l e Ser 885 890 895 Al a Ty r Leu Leu Ser Lys As n As n Al a Ile Gl u Pr o Ar g Ser Phe Ser 900 905 910 Gl n As n Gl y Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 915 920 925 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser 930 935 940 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser 945 950 955 960

   Page 344

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   Gl y Ser Gl u Thr Pr o Gl y Thr Ser 965

   Pr o Gl y Thr Ser Thr Gl u Pr o Ser 980

   Al a Gl y Ser Pr o Thr Ser Thr Gl u 995 1000

   Pr o Gl u 1010 Ser Gl y Pr o Gl y Ser 1015 Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a 1025 1030 Ser Pr o Al a Gl y Ser Pr o Thr Ser 1040 1045 Gl y Ser Pr o Thr Ser Thr Gl u Gl u 1055 1060 Gl u Gl y Ser Al a Pr o Gl y Thr Ser 1070 1075 Gl y Pr o Gl y Thr Ser Gl u Ser Al a

   97047_1 Gl u Ser Al a Thr Pr o Gl u Ser Gl y 970 975

   Gl u Gl y Ser Al a Pr o Gl y Ser Pr o 985 990

   u Gl y Thr Ser Gl u Ser Al a Thr

   1005 Pr o Al a Thr Ser 1020 Gl y Ser Gl u Thr Pr o Gl u Ser 1035 Gl y Pr o Gl y Thr Gl u Gl u Gl y 1050 Ser Pr o Al a Gl y Thr Ser Thr 1065 Gl u Pr o Ser Gl u Ser Al a Thr 1080 Pr o Gl u Ser Thr Pr o Gl u Ser Gl y Pr o Gl y

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   1085 1090 97047_1 1095 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 1100 1105 1110 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser 1115 1120 1125 Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr 1130 1135 1140 Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 1145 1150 1155 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o 1160 1165 1170 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr 1175 1180 1185 Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser 1190 1195 1200

   Al a Pr o

   Al a Ser Ser

   Pr o Pr o

   Val Leu Lys Ar g Hi s

   Gl n Al a Gl u

   1205

   1210

   1215

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   97047_1

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   I l e Thr Ar g Thr Thr Leu Gl n 1225 Ser As p Gl n Gl u Gl u 1230 I l e As p Ty r 1220 As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u As p Phe As p I l e 1235 1240 1245 Ty r As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys 1250 1255 1260 Thr Ar g Hi s Ty r Phe Ile Al a Al a Val Gl u Ar g Leu Tr p As p Ty r 1265 1270 1275 Gl y Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser 1280 1285 1290 Gl y Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr 1295 1300 1305 As p Gl y Ser Phe Thr Gl n Pr o Leu Ty r Ar g Gl y Gl u Leu As n Gl u 1310 1315 1320 Hi s Leu Gl y Leu Leu Gl y Pr o Ty r Ile Ar g Al a Gl u Val Gl u As p 1325 1330 1335 As n I l e Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Ty r Ser

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   1340 1345 1350

   Phe Tyr Ser Ser Leu Ile Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl ^y 1355 1360 1365 Al a Gl u Pr o Ar g Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Th r 1370 1375 1380 Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u 1385 1390 1395 Phe As p Cys Lys Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u 1400 1405 1410 Lys As p Val Hi s Ser Gl y Leu Ile Gl y Pr o Leu Leu Val Cys Hi s 1415 1420 1425 Thr As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n 1430 1435 1440 Gl u Phe Al a Leu Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr ^p 1445 1450 1455 Tyr Phe Thr Gl u As n Met Gl u Ar g As n Cys Ar g Al a Pr o Cys As n

   1460 1465 1470

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   I l e Gl n 1475 Met Gl u As p Pr o Thr 1480 Phe Lys Gl u As n Ty r 1485 Ar g Phe Hi s Al a I l e As n Gl y Tyr Ile Met As p Thr Leu Pr o Gl y Leu Val Met 1490 1495 1500 Al a Gl n Asp Gl n Ar g Ile Ar g Tr p Ty r Leu Leu Ser Met Gl y Ser 1505 1510 1515 As n Gl u As n Ile Hi s Ser Ile Hi s Phe Ser Gl y Hi s Val Phe Thr 1520 1525 1530 Val Ar g Lys Lys Gl u Gl u Ty r Lys Met Al a Leu Ty r As n Leu Ty r 1535 1540 1545 Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y 1550 1555 1560 I l e Tr p Ar g Val Gl u Cys Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y 1565 1570 1575 Met Ser Thr Leu Phe Leu Val Ty r Ser As n Lys Cy s Gl n Thr Pr o 1580 1585 1590 Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n I l e Thr Al a

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   97047_1

   1595 1600 1605

   Ser Gl y 1610 Gl n Ty r Gl y Gl n Tr p 1615 Al a Pr o Lys Leu Al a 1620 Ar g Leu Hi s Ty r Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser 1625 1630 1635 Tr p I l e Lys Val As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y I l e 1640 1645 1650 Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r I l e Ser 1655 1660 1665 Gl n Phe I l e Ile Met Ty r Ser Leu As p Gl y Lys Lys Tr p Gl n Thr 1670 1675 1680 Ty r Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n 1685 1690 1695 Val As p Ser Ser Gl y Ile Lys Hi s As n Ile Phe As n Pr o Pr o I l e 1700 1705 1710 I l e Al a Ar g Ty r Ile Ar g Leu Hi s Pr o Thr Hi s Ty r Ser I l e Ar g

   1715 1720 1725

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   Ser Thr 1730 Leu Ar g Met Gl u Leu 1735 Met Gl y Cys As p Leu 1740 As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n 1745 1750 1755 I l e Thr Al a Ser Ser Tyr Phe Thr As n Met Phe Al a Thr Tr p Ser 1760 1765 1770 Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p 1775 1780 1785 Ar g Pr o Gl n Val As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe 1790 1795 1800 Gl n Lys Thr Met Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys 1805 1810 1815 Ser Leu Leu Thr Ser Met Tyr Val Lys Gl u Phe Leu I l e Ser Ser 1820 1825 1830 Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys 1835 1840 1845 Val Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val

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   1850 1855 97047 _1 1860 As n Ser Leu As p Pr o Pr o Leu Leu Thr Ar g Tyr Leu Ar g I l e Hi s 1865 1870 1875 Pr o Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu 1880 1885 1890 Gl y Cys Gl u Al a Gl n As p Leu Tyr As p Lys Thr Hi s Thr Cys Pr o 1895 1900 1905 Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu 1910 1915 1920 Phe Pr o Pr o Lys Pr o Lys As p Thr Leu Met Ile Ser Ar g Thr Pr o 1925 1930 1935 Gl u Val Thr Cys Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u 1940 1945 1950 Val Lys Phe As n Tr p Tyr Val As p Gl y Val Gl u Val Hi s As n Al a 1955 1960 1965 Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr As n Ser Thr Tyr Ar g Val 1970 1975 1980

   Page 352

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   Val Ser 1985 Val Leu Thr Val Leu 1990 Hi s Gl n As p Tr p Leu 1995 As n Gl y Lys Gl u Ty r Lys Cy s Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o I l e 2000 2005 2010 Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n 2015 2020 2025 Val Ty r Thr Leu Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n 2030 2035 2040 Val Ser Leu Thr Cy s Leu Val Lys Gl y Phe Ty r Pr o Ser As p I l e 2045 2050 2055 Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Ty r Lys 2060 2065 2070 Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe Phe Leu Ty r 2075 2080 2085 Ser Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val 2090 2095 2100 Phe Ser Cy s Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s Ty r Thr

   Page 353

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   2105 2110 2115

   Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y Lys 2120 2125 <210> 112 <211> 2020 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN FVI I I 266 pr ot ei n sequence <400> 112

   Met Gl n I l e Gl u Le u Ser Thr Cys Phe Phe Leu Cys Leu Leu Ar g Phe 1 5 10 15 Cys Phe Ser Al a Th r Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Gl y Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 35 40 45 Ser Thr Gl u Gl u Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 50 55 60 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Al a Ser Ser Ser

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   65 70 75 80

   As p Leu Gl y Gl u Leu 85 Pr o Val As p Al a Ar g 90 Phe Pr o Pr o Ar g Val 95 Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val Ty r Lys Lys Thr Leu Phe 100 105 110 Val Gl u Phe Thr As p Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o 115 120 125 Tr p Met Gl y Leu Leu Gl y Pr o Thr Ile Gl n Al a Gl u Val Ty r As p Thr 130 135 140 Val Val I l e Thr Leu Lys As n Met Al a Ser Hi s Pr o Val Ser Leu Hi s 145 150 155 160 Al a Val Gl y Val Ser Ty r Tr p Lys Al a Ser Gl u Gl y Al a Gl u Ty r As p 165 170 175 As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys Val Phe Pr o Gl y 180 185 190 Gl y Ser Hi s Thr Ty r Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met 195 200 205

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   Al a Ser 210 As p Pr o Leu Cys Leu 215 Thr Tyr Ser Tyr Leu 220 Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cys 225 230 235 240 Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr Leu Hi s Lys Phe 245 250 255 I l e Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser Tr p Hi s Ser Gl u 260 265 270 Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a Ser Al a Ar g Al a 275 280 285 Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr Val As n Ar g Ser Leu Pr o 290 295 300 Gl y Leu I l e Gl y Cys Hi s Ar g Lys Ser Val Tyr Tr p Hi s Val I l e Gl y 305 310 315 320 Met Gl y Thr Thr Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr 325 330 335 Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u I l e Ser Pr o I l e

   Page 356

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   970 47_1 340 345 350 Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu 355 360 365 Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y Met Gl u Al a Tyr 370 375 380 Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n 385 390 395 400 As n Gl u Gl u Al a Gl u As p Tyr As p As p As p Leu Thr As p Ser Gl u Met 405 410 415 As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser Phe I l e Gl n I l e 420 425 430 Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Tyr I l e Al a 435 440 445 Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o Leu Val Leu Al a Pr o As p 450 455 460 As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n As n Gl y Pr o Gl n Ar g I l e 465 470 475 480

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   Gl y Ar g Lys Tyr Lys 485 Lys Val Ar g Phe Met 490 Al a Tyr Thr As p Gl u 495 Thr Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y I l e Leu Gl y Pr o 500 505 510 Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu Leu Ile I l e Phe Lys As n 515 520 525 Gl n Al a Ser Ar g Pr o Tyr As n Ile Tyr Pr o Hi s Gl y I l e Thr As p Val 530 535 540 Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys 545 550 555 560 As p Phe Pr o I l e Leu Pr o Gl y Gl u Ile Phe Lys Tyr Lys Tr p Thr Val 565 570 575 Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g Cys Leu Thr Ar g 580 585 590 Tyr Tyr Ser Ser Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu 595 600 605 I l e Gl y Pr o Leu Leu Ile Cys Tyr Lys Gl u Ser Val As p Gl n Ar g Gl y

   Page 358

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   97047_1

   610 615 620

   As n 625 Gl n I l e Met Ser As p 630 Lys Ar g As n Val Ile 635 Leu Phe Ser Val Phe 640 As p Gl u As n Ar g Ser Tr p Tyr Leu Thr Gl u As n Ile Gl n Ar g Phe Leu 645 650 655 Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser 660 665 670 As n I l e Met Hi s Ser Ile As n Gl y Tyr Val Phe As p Ser Leu Gl n Leu 675 680 685 Ser Val Cys Leu Hi s Gl u Val Al a Tyr Tr p Tyr Ile Leu Ser I l e Gl y 690 695 700 Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y Tyr Thr Phe Lys 705 710 715 720 Hi s Lys Met Val Tyr Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y 725 730 735 Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu Tr p I l e Leu Gl y 740 745 750

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   Cys Hi s Asn Ser As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys 755 760 76 5 Val Ser Ser Cys As p Lys As n Thr Gl y As p Tyr Tyr Gl u As p Ser Tyr 770 775 780 Gl u Asp I l e Ser Al a Tyr Leu Leu Ser Lys As n As n Al a I l e Gl u Pr o 785 790 795 800 Arg Ser Phe Ser Gl n As n Gl y Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr 805 810 815 Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl ^y Ser Gl u Thr 820 825 830 Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u 835 840 84 5 Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr 850 855 860 Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a 865 870 875 880 Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser

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   Gl u Ser Al a Thr 900 885 Pr o 890 Ser 895 Ser Gl u Ser Gl y Pr o 905 Gl y Gl u Pr o Al a Thr 910 Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 915 920 925 Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o 930 935 940 Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser 945 950 955 960 Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 965 970 975 Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser 980 985 990 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser 995 1000 1005 Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u 1010 1015 1020

   Page 361

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   Thr Pr o 1025 Gl y Ser Pr o Al a Gl y 1030 Ser Pr o Thr Ser Thr 1035 Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr 1040 1045 1050 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o Al a Thr Ser 1055 1060 1065 Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser 1070 1075 1080 Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Al a 1085 1090 1095 Ser Ser Pr o Pr o Val Leu Lys Ar g Hi s Gl n Al a Gl u I l e Thr Ar g 1100 1105 1110 Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u Ile As p Tyr As p As p Thr 1115 1120 1125 I l e Ser Val Gl u Met Lys Lys Gl u As p Phe As p Ile Tyr As p Gl u 1130 1135 1140 Asp Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s

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   1145 1150 97047 _1 1155 Tyr Phe I l e Al a Al a Val Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser 1160 1165 1170 Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val 1175 1180 1185 Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y Ser 1190 1195 1200 Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y 1205 1210 1215 Leu Leu Gl y Pr o Tyr Ile Ar g Al a Gl u Val Gl u As p As n I l e Met 1220 1225 1230 Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser 1235 1240 1245 Ser Leu I l e Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o 1250 1255 1260 Ar g Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p 1265 1270 1275

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   Lys Val 1280 Gl n Hi s Hi s Met Al a 1285 Pr o Thr Lys As p Gl u 1290 Phe As p Cys Lys Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u Lys As p Val 1295 1300 1305 Hi s Ser Gl y Leu Ile Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr 1310 1315 1320 Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a 1325 1330 1335 Leu Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p Tyr Phe Thr 1340 1345 1350 Gl u As n Met Gl u Ar g As n Cys Ar g Al a Pr o Cys As n I l e Gl n Met 1355 1360 1365 Gl u As p Pr o Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s Al a I l e As n 1370 1375 1380 Gl y Tyr I l e Met As p Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p 1385 1390 1395 Gl n Ar g I l e Ar g Tr p Tyr Leu Leu Ser Met Gl y Ser As n Gl u As n

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   1400 1405 97047 _1 1410 I l e Hi s Ser Ile Hi s Phe Ser Gl y Hi s Val Phe Thr Val Ar g Lys 1415 1420 1425 Lys Gl u Gl u Ty r Lys Met Al a Leu Ty r As n Leu Ty r Pr o Gl y Val 1430 1435 1440 Phe Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y I l e Tr p Ar g 1445 1450 1455 Val Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr 1460 1465 1470 Leu Phe Leu Val Ty r Ser As n Lys Cy s Gl n Thr Pr o Leu Gl y Met 1475 1480 1485 Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n 1490 1495 1500 Ty r Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s Ty r Ser Gl y 1505 1510 1515 Ser I l e As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p I l e Lys 1520 1525 1530

   Page 365

   97047_1

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   Val As p 1535 Leu Leu Al a Pr o Met 1540 Ile Ile Hi s Gl y Ile 1545 Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r Ile Ser Gl n Phe I l e 1550 1555 1560 I l e Met Ty r Ser Leu As p Gl y Lys Lys Tr p Gl n Thr Ty r Ar g Gl y 1565 1570 1575 As n Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n Val As p Ser 1580 1585 1590 Ser Gl y I l e Lys Hi s As n Ile Phe As n Pr o Pr o Ile I l e Al a Ar g 1595 1600 1605 Ty r I l e Ar g Leu Hi s Pr o Thr Hi s Ty r Ser Ile Ar g Ser Thr Leu 1610 1615 1620 Ar g Met Gl u Leu Met Gl y Cy s As p Leu As n Ser Cy s Ser Met Pr o 1625 1630 1635 Leu Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n I l e Thr Al a 1640 1645 1650 Ser Ser Ty r Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys

   Page 366

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   1655 1660 97047 _1 1665 Al a Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n 1670 1675 1680 Val As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr 1685 1690 1695 Met Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys Ser Leu Leu 1700 1705 1710 Thr Ser Met Tyr Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p 1715 1720 1725 Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val 1730 1735 1740 Phe Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu 1745 1750 1755 As p Pr o Pr o Leu Leu Thr Ar g Tyr Leu Ar g Ile Hi s Pr o Gl n Ser 1760 1765 1770 Tr p Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu Gl y Cys Gl u 1775 1780 1785

   Page 367

   97047_1

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   Al a Gl n 1790 As p Leu Ty r As p Lys 1795 Thr Hi s Thr Cy s Pr o 1800 Pr o Cy s Pr o Al a Pr o Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o 1805 1810 1815 Lys Pr o Lys As p Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr 1820 1825 1830 Cy s Val Val Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe 1835 1840 1845 As n Tr p Ty r Val As p Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys 1850 1855 1860 Pr o Ar g Gl u Gl u Gl n Ty r As n Ser Thr Ty r Ar g Val Val Ser Val 1865 1870 1875 Leu Thr Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Ty r Lys 1880 1885 1890 Cy s Lys Val Ser As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr 1895 1900 1905 I l e Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Ty r Thr

   Page 368

   97047_1

   2018203206 08 May 2018

   1910 1915 1920 Leu Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n Val Ser Leu 1925 1930 1935 Thr Cys Leu Val Lys Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u 1940 1945 1950 Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o 1955 1960 1965 Pr o Val Leu As p Ser As p Gl y Ser Phe Phe Leu Tyr Ser Lys Leu 1970 1975 1980 Thr Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser Cys 1985 1990 1995 Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser 2000 2005 2010 Leu Ser Leu Ser Pr o Gl y Lys

   2015 2020 <210> 113 <211> 2056 <212> PRT

   Page 369

   2018203206 08 May 2018

   97047_1 <213> Art i f i ci al Sequence <220>

   <223> pSYN FVI I I 267 pr ot ei n sequence <400> 113

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cy s Phe Phe 10 Leu Cy s Leu Leu Ar g 15 Phe Cy s Phe Ser Al a Thr Ar g Ar g Ty r Ty r Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Ty r Met Gl n Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u 35 40 45 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 50 55 60 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 65 70 75 80 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 85 90 95 Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 100 105 110

   Page 370

   97047_1

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   Al a Ser Ser 115 Ser As p Leu Gl y Gl u 120 Leu Pr o Val As p Al a 125 Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val Tyr Lys 130 135 140 Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e Al a Lys 145 150 155 160 Pr o Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr I l e Gl n Al a Gl u 165 170 175 Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser Hi s Pr o 180 185 190 Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y 195 200 205 Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys 210 215 220 Val Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu Lys Gl u 225 230 235 240 As n Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser Tyr Leu

   Page 371

   2018203206 08 May 2018

   97047_1

   245 250 255

   Ser Hi s Val Asp Leu Val 260 Lys As p Leu 265 As n Ser Gl y Leu I l e 270 Gl y Al a Leu Leu Val Cy s Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr 275 280 285 Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser 290 295 300 Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a 305 310 315 320 Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Ty r Val As n 325 330 335 Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cy s Hi s Ar g Lys Ser Val Ty r Tr p 340 345 350 Hi s Val I l e Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e Phe Leu 355 360 365 Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u 370 375 380

   Page 372

   97047_1

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   I l e 385 Ser Pr o I l e Thr Phe 390 Leu Thr Al a Gl n Thr 395 Leu Leu Met As p Leu 400 Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y 405 410 415 Met Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu 420 425 430 Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Tyr As p As p As p Leu Thr 435 440 445 As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser 450 455 460 Phe I l e Gl n I l e Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val 465 470 475 480 Hi s Tyr I l e Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o Leu Val 485 490 495 Leu Al a Pr o As p As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n As n Gl y 500 505 510 Pr o Gl n Ar g I l e Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met Al a Tyr

   Page 373

   2018203206 08 May 2018

   97047_1

   515 520 525

   Thr As p 530 Gl u Thr Phe Lys Thr 535 Ar g Gl u Al a Ile Gl n 540 Hi s Gl u Ser Gl y I l e Leu Gl y Pr o Leu Leu Ty r Gl y Gl u Val Gl y As p Thr Leu Leu I l e 545 550 555 560 I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Ty r As n Ile Ty r Pr o Hi s Gl y 565 570 575 I l e Thr As p Val Ar g Pr o Leu Ty r Ser Ar g Ar g Leu Pr o Lys Gl y Val 580 585 590 Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe Lys Ty r 595 600 605 Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g 610 615 620 Cy s Leu Thr Ar g Ty r Ty r Ser Ser Phe Val As n Met Gl u Ar g As p Leu 625 630 635 640 Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cy s Ty r Lys Gl u Ser Val 645 650 655

   Page 374

   97047_1

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   As p Gl n Ar g Gl y 660 As n Gl n Ile Met Ser 665 As p Lys Ar g As n Val 670 I l e Leu Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Tyr Leu Thr Gl u As n I l e 675 680 685 Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u 690 695 700 Phe Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Tyr Val Phe As p 705 710 715 720 Ser Leu Gl n Leu Ser Val Cys Leu Hi s Gl u Val Al a Tyr Tr p Tyr I l e 725 730 735 Leu Ser I l e Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y 740 745 750 Tyr Thr Phe Lys Hi s Lys Met Val Tyr Gl u As p Thr Leu Thr Leu Phe 755 760 765 Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu 770 775 780 Tr p I l e Leu Gl y Cys Hi s As n Ser As p Phe Ar g As n Ar g Gl y Met Thr

   Page 375

   2018203206 08 May 2018

   97047_1

   785 790 795 800

   Al a Leu Leu Lys Val Ser Ser Cys As p Lys As n Thr Gl y As p Tyr Tyr 805 810 815 Gl u As p Ser Tyr Gl u As p Ile Ser Al a Tyr Leu Leu Ser Lys As n As n 820 825 830 Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Gl y Al a Pr o Gl y Thr Ser 835 840 845 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser 850 855 860 Gl ^y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 86 5 870 875 880 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser 885 890 895 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser 900 905 910 Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u 915 920 925

   Page 376

   97047_1

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   Gl u Gl y 930 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 935 940 Gl y Ser Gl u Pr o 945 Al a Thr Ser Gl y Ser 950 Gl u Thr Pr o Gl y Thr Ser Gl u 955 Ser Al a Thr 960 Pr o Gl u Ser Gl y Pr o 965 Gl y Ser Pr o Al a Gl y Ser Pr o Thr 970 Ser Thr 975 Gl u Gl u Gl y Ser Pr o 980 Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u 985 Gl y 990 Thr Ser Thr Gl u Pr o 995 Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr 1000 1005 Pr o Gl u 1010 Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr 1015 1020 Pr o Gl u Ser Gl y Pr o 1025 Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser 1030 1035 Gl y Pr o Gl y Ser Gl u 1040 Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 1045 1050 Ser Gl u Pr o

   Al a Thr

   Ser Gl y Ser Gl u Thr

   Pr o Gl y Ser Pr o Al a

   Gl y Ser Pr o

   Page 377

   2018203206 08 May 2018

   1055 1060 97047 _1 1065 Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser 1070 1075 1080 Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 1085 1090 1095 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u 1100 1105 1110 Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser 1115 1120 1125 Gl u Gl y Ser Al a Pr o Al a Ser Ser Pr o Pr o Val Leu Lys Ar g Hi s 1130 1135 1140 Gl n Al a Gl u Ile Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u 1145 1150 1155 I l e As p Tyr As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u As p 1160 1165 1170 Phe As p I l e Tyr As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe 1175 1180 1185

   Page 378

   97047_1

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   Gl n Lys 1190 Lys Thr Ar g Hi s Tyr 1195 Phe Ile Al a Al a Val 1200 Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g 1205 1210 1215 Al a Gl n Ser Gl y Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n 1220 1225 1230 Gl u Phe Thr As p Gl y Ser Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u 1235 1240 1245 Leu As n Gl u Hi s Leu Gl y Leu Leu Gl y Pr o Tyr Ile Ar g Al a Gl u 1250 1255 1260 Val Gl u As p As n Ile Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g 1265 1270 1275 Pr o Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Gl u Gl u As p Gl n 1280 1285 1290 Ar g Gl n Gl y Al a Gl u Pr o Ar g Lys As n Phe Val Lys Pr o As n Gl u 1295 1300 1305 Thr Lys Thr Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met Al a Pr o Thr

   Page 379

   2018203206 08 May 2018

   1310 1315 97047 _1 1320 Lys As p Gl u Phe As p Cys Lys Al a Tr p Al a Tyr Phe Ser As p Val 1325 1330 1335 As p Leu Gl u Lys As p Val Hi s Ser Gl y Leu Ile Gl y Pr o Leu Leu 1340 1345 1350 Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val 1355 1360 1365 Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr Ile Phe As p Gl u Thr 1370 1375 1380 Lys Ser Tr p Tyr Phe Thr Gl u As n Met Gl u Ar g As n Cys Ar g Al a 1385 1390 1395 Pr o Cys As n Ile Gl n Met Gl u As p Pr o Thr Phe Lys Gl u As n Tyr 1400 1405 1410 Ar g Phe Hi s Al a Ile As n Gl y Tyr Ile Met As p Thr Leu Pr o Gl y 1415 1420 1425 Leu Val Met Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr Leu Leu Ser 1430 1435 1440

   Page 380

   97047_1

   2018203206 08 May 2018

   Met Gl y 1445 Ser As n Gl u As n Ile 1450 Hi s Ser Ile Hi s Phe 1455 Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Ty r Lys Met Al a Leu Ty r 1460 1465 1470 As n Leu Ty r Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser 1475 1480 1485 Lys Al a Gl y Ile Tr p Ar g Val Gl u Cy s Leu Ile Gl y Gl u Hi s Leu 1490 1495 1500 Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Ty r Ser As n Lys Cy s 1505 1510 1515 Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n 1520 1525 1530 I l e Thr Al a Ser Gl y Gl n Ty r Gl y Gl n Tr p Al a Pr o Lys Leu Al a 1535 1540 1545 Ar g Leu Hi s Ty r Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u 1550 1555 1560 Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o Met I l e I l e

   Page 381

   2018203206 08 May 2018

   1565 1570 97047 _1 1575 Hi s Gl y I l e Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu 1580 1585 1590 Tyr I l e Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p Gl y Lys Lys 1595 1600 1605 Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe 1610 1615 1620 Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n I l e Phe As n 1625 1630 1635 Pr o Pr o I l e Ile Al a Ar g Tyr Ile Ar g Leu Hi s Pr o Thr Hi s Tyr 1640 1645 1650 Ser I l e Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y Cys As p Leu 1655 1660 1665 As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a I l e Ser 1670 1675 1680 As p Al a Gl n Ile Thr Al a Ser Ser Tyr Phe Thr As n Met Phe Al a 1685 1690 1695

   Page 382

   97047 1

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   Thr Tr p 1700 Ser Pr o Ser Lys Al a 1705 Ar g Leu Hi s Leu Gl n 1710 Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u Tr p Leu Gl n 1715 1720 1725 Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val Thr Thr Gl n 1730 1735 1740 Gl y Val Lys Ser Leu Leu Thr Ser Met Ty r Val Lys Gl u Phe Leu 1745 1750 1755 I l e Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n 1760 1765 1770 As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr 1775 1780 1785 Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr Ar g Ty r Leu 1790 1795 1800 Ar g I l e Hi s Pr o Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met 1805 1810 1815 Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p Leu Ty r As p Lys Thr Hi s

   Page 383

   2018203206 08 May 2018

   1820 1825 97047 _1 1830 Thr Cys Pr o Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y Gl y Pr o Ser 1835 1840 1845 Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu Met I l e Ser 1850 1855 1860 Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val Ser Hi s Gl u 1865 1870 1875 As p Pr o Gl u Val Lys Phe As n Tr p Tyr Val As p Gl y Val Gl u Val 1880 1885 1890 Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr As n Ser Thr 1895 1900 1905 Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n As p Tr p Leu 1910 1915 1920 As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys Al a Leu Pr o 1925 1930 1935 Al a Pr o I l e Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g 1940 1945 1950

   Page 384

   97047_1

   2018203206 08 May 2018

   Gl u Pr o 1955 Gl n Val Tyr Thr Leu 1960 Pr o Pr o Ser Ar g As p 1965 Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y Phe Tyr Pr o 1970 1975 1980 Ser As p I l e Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n 1985 1990 1995 As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe 2000 2005 2010 Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n 2015 2020 2025 Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a Leu Hi s As n 2030 2035 2040 Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y Lys 2045 2050 2055

   <210> 114 <211> 1834 <212> PRT <213> Art i f i ci al Sequence

   Page 385

   2018203206 08 May 2018

   97047_1 <220>

   <223> pSYN FVI I I 268 protei n sequence <400> 114

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cy s Phe Phe 10 Leu Cy s Leu Leu Ar g 15 Phe Cy s Phe Ser Al a Thr Ar g Ar g Ty r Ty r Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Ty r Met Gl n Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u 35 40 45 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 50 55 60 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 65 70 75 80 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u 85 90 95 Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 100 105 110 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser

   Page 386

   2018203206 08 May 2018

   97047_1

   115 120 125

   Al a Thr 130 Pr o Gl u Ser Gl y Pr o 135 Gl y Ser Gl u Pr o Al a 140 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 145 150 155 160 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 165 170 175 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Al a Ser Ser Ser As p Leu Gl y Gl u 180 185 190 Leu Pr o Val As p Al a Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o 195 200 205 Phe As n Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Gl u Phe Thr 210 215 220 As p Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu 225 230 235 240

   Leu Gl y Pr o Thr

   I l e Gl n Al a Gl u

   Val

   Tyr Asp Thr Val

   Val

   I l e Thr

   245

   250

   255

   Page 387

   97047_1

   2018203206 08 May 2018

   Leu Lys As n Met 260 Al a Ser Hi s Pr o Val 265 Ser Leu Hi s Al a Val 270 Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser 275 280 285 Gl n Ar g Gl u Lys Gl u As p As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr 290 295 300 Tyr Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o 305 310 315 320 Leu Cys Leu Thr Tyr Ser Tyr Leu Ser Hi s Val As p Leu Val Lys As p 325 330 335 Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser 340 345 350 Leu Al a Lys Gl u Lys Thr Gl n Thr Leu Hi s Lys Phe I l e Leu Leu Phe 355 360 365 Al a Val Phe As p Gl u Gl y Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser 370 375 380 Leu Met Gl n As p Ar g As p Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met

   Page 388

   2018203206 08 May 2018

   97047_1

   385 390 395 400

   Hi s Thr Val As n Gl y 405 Ty r Val As n Ar g Ser 410 Leu Pr o Gl y Leu I l e 415 Gl y Cy s Hi s Ar g Lys Ser Val Ty r Tr p Hi s Val Ile Gl y Met Gl y Thr Thr 420 425 430 Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g 435 440 445 As n Hi s Ar g Gl n Al a Ser Leu Gl u Ile Ser Pr o Ile Thr Phe Leu Thr 450 455 460 Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu Leu Phe Cy s Hi s 465 470 475 480 I l e Ser Ser Hi s Gl n Hi s As p Gl y Met Gl u Al a Ty r Val Lys Val As p 485 490 495 Ser Cy s Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a 500 505 510 Gl u As p Ty r As p As p As p Leu Thr As p Ser Gl u Met As p Val Val Ar g 515 520 525

   Page 389

   97047_1

   2018203206 08 May 2018

   Phe Asp Asp Asp Asn Ser 530 Pr o 535 Ser Phe Ile Gl n Ile 540 Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Ty r Ile Al a Al a Gl u Gl u Gl u 545 550 555 560 As p Tr p As p Ty r Al a Pr o Leu Val Leu Al a Pr o As p As p Ar g Ser Ty r 565 570 575 Lys Ser Gl n Ty r Leu As n As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Ty r 580 585 590 Lys Lys Val Ar g Phe Met Al a Ty r Thr As p Gl u Thr Phe Lys Thr Ar g 595 600 605 Gl u Al a I l e Gl n Hi s Gl u Ser Gl y Ile Leu Gl y Pr o Leu Leu Ty r Gl y 610 615 620 Gl u Val Gl y As p Thr Leu Leu Ile Ile Phe Lys As n Gl n Al a Ser Ar g 625 630 635 640 Pr o Ty r As n I l e Ty r Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Ty r 645 650 655 Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o I l e

   Page 390

   2018203206 08 May 2018

   660 665 97047_1 670 Leu Pr o Gl y Gl u Ile Phe Lys Ty r Lys Tr p Thr Val Thr Val Gl u As p 675 680 685 Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s Leu Thr Ar g Ty r Ty r Ser Ser 690 695 700 Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu 705 710 715 720 Leu I l e Cy s Ty r Lys Gl u Ser Val As p Gl n Ar g Gl y As n Gl n I l e Met 725 730 735 Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe As p Gl u As n Ar g 740 745 750 Ser Tr p Ty r Leu Thr Gl u As n Ile Gl n Ar g Phe Leu Pr o As n Pr o Al a 755 760 765 Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser As n I l e Met Hi s 770 775 780 Ser I l e As n Gl y Ty r Val Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu 785 790 795 800

   Page 391

   97047_1

   2018203206 08 May 2018

   Hi s Gl u Val Al a Tyr 805 Trp Tyr I l e Leu Ser 810 Ile Gl y Al a Gl n Thr 815 As p Phe Leu Ser Val Phe Phe Ser Gl y Tyr Thr Phe Lys Hi s Lys Met Val 820 825 830 Tyr Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe 835 840 845 Met Ser Met Gl u As n Pr o Gl y Leu Tr p Ile Leu Gl y Cys Hi s As n Ser 850 855 860 As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cys 865 870 875 880 As p Lys As n Thr Gl y As p Tyr Tyr Gl u As p Ser Tyr Gl u As p I l e Ser 885 890 895 Al a Tyr Leu Leu Ser Lys As n As n Al a Ile Gl u Pr o Ar g Ser Phe Ser 900 905 910 Gl n As n Pr o Pr o Val Leu Lys Ar g Hi s Gl n Al a Gl u I l e Thr Ar g Thr 915 920 925 Thr Leu Gl n Ser As p Gl n Gl u Gl u Ile As p Tyr As p As p Thr I l e Ser

   Page 392

   97047_1

   2018203206 08 May 2018

   930 935 940 Val Gl u Met Lys Lys Gl u As p Phe As p Ile Tyr As p Gl u As p Gl u As n 945 950 955 960 Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s Tyr Phe I l e Al a 965 970 975 Al a Val Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser Ser Pr o Hi s Val 980 985 990 Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o Gl n Phe Ly s Ly s Val 995 1000 10C 5

   Val Phe

   Gl n Gl u Phe Thr As p

   Gl y Ser Phe Thr Gl n

   Pro Leu Tyr

   1010

   1015

   1020

   Ar g Gl y

   Gl u Leu Asn Gl u

   Hi s

   Leu Gl y Leu Leu Gl y

   Pro Tyr I l e

   1025

   1030

   1035

   Ar g Al a

   Glu Val Glu Asp Asn

   I l e Met Val

   Thr Phe

   Ar g As n Gl n

   1040

   1045

   1050

   Al a Ser

   Arg Pro Tyr Ser Phe

   Tyr Ser Ser Leu I l e

   Ser Tyr Gl u

   1055

   1060

   1065

   Page 393

   97047_1

   2018203206 08 May 2018

   Gl u As p 1070 Gl n Ar g Gl n Gl y Al a 1075 Gl u Pr o Ar g Lys As n 1080 Phe Val Lys Pr o As n Gl u Thr Lys Thr Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met 1085 1090 1095 Al a Pr o Thr Lys As p Gl u Phe As p Cys Lys Al a Tr p Al a Tyr Phe 1100 1105 1110 Ser As p Val As p Leu Gl u Lys As p Val Hi s Ser Gl y Leu I l e Gl y 1115 1120 1125 Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y 1130 1135 1140 Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe 1145 1150 1155 As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u As n Met Gl u Ar g As n 1160 1165 1170 Cys Ar g Al a Pr o Cys As n Ile Gl n Met Gl u As p Pr o Thr Phe Lys 1175 1180 1185 Gl u As n Tyr Ar g Phe Hi s Al a Ile As n Gl y Tyr Ile Met As p Thr

   Page 394

   2018203206 08 May 2018

   97047_1

   1190 1195 1200

   Leu Pr o 1205 Gl y Leu Val Met Al a 1210 Gl n As p Gl n Ar g Ile 1215 Ar g Tr p Tyr Leu Leu Ser Met Gl y Ser As n Gl u As n Ile Hi s Ser I l e Hi s Phe 1220 1225 1230 Ser Gl y Hi s Val Phe Thr Val Arg Lys Lys Gl u Gl u Tyr Lys Met 1235 1240 1245 Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe Gl u Thr Val Gl u Met 1250 1255 1260 Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys Leu I l e Gl y 1265 1270 1275 Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Tyr Ser 1280 1285 1290 As n Lys Cys Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g 1295 1300 1305

   As p Phe

   Gl n I l e Thr

   Al a Ser

   Gly Gln Tyr Gly Gln

   Tr p Al a Pr o

   1310

   1315

   1320

   Page 395

   97047_1

   2018203206 08 May 2018

   Lys Leu 1325 Al a Ar g Leu Hi s Tyr 1330 Ser Gl y Ser Ile As n 1335 Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o 1340 1345 1350 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe 1355 1360 1365 Ser Ser Leu Tyr Ile Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p 1370 1375 1380 Gl y Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu 1385 1390 1395 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n 1400 1405 1410 I l e Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr Ile Ar g Leu Hi s Pr o 1415 1420 1425 Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g Met Gl u Leu Met Gl y 1430 1435 1440 Cys As p Leu As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys

   Page 396

   2018203206 08 May 2018

   1445 1450 97047 _1 1455 Al a I l e Ser As p Al a Gl n Ile Thr Al a Ser Ser Tyr Phe Thr As n 1460 1465 1470 Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n 1475 1480 1485 Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u 1490 1495 1500 Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1505 1510 1515 Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1520 1525 1530 Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu 1535 1540 1545 Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p 1550 1555 1560 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1565 1570 1575

   Page 397

   97047 1

   2018203206 08 May 2018

   Arg Tyr Leu Ar g Ile Hi s Pr o 1585 Gl n Ser Tr p Val Hi s 1590 Gl n I l e Al a 1580 Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p Leu Ty r As p 1595 1600 1605 Lys Thr Hi s Thr Cys Pr o Pr o Cy s Pr o Al a Pr o Gl u Leu Leu Gl y 1610 1615 1620 Gly Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1625 1630 1635 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cy s Val Val Val As p Val 1640 1645 1650 Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Ty r Val As p Gl y 1655 1660 1665 Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Ty r 1670 1675 1680 Asn Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n 1685 1690 1695 Asp Tr p Leu As n Gl y Lys Gl u Ty r Lys Cy s Lys Val Ser As n Lys

   Page 398

   2018203206 08 May 2018

   97047_1

   1700 1705 1710

   Al a Leu 1715 Pr o Al a Pr o Ile Gl u 1720 Lys Thr Ile Ser Lys 1725 Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p 1730 1735 1740 Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y 1745 1750 1755 Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n 1760 1765 1770 Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p 1775 1780 1785 Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g 1790 1795 1800 Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a 1805 1810 1815 Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y

   1820 1825 1830

   Page 399

   97047_1

   2018203206 08 May 2018

   Lys <210> 115 <211> 1762 <212> PRT <213> Artificial Sequence <220>

   <223> pSYN FVI I I 269 pr ot ei n sequence <400> 115

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cys Phe Phe 10 Leu Cys Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u 35 40 45 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y 50 55 60 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a 65 70 75 80

   Page 400

   97047_1

   2018203206 08 May 2018

   Thr Ser Gl y Ser Gl u 85 Thr Pr o Gl y Thr Ser 90 Gl u Ser Al a Thr Pr o 95 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 100 105 110 Al a Ser Ser Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g Phe Pr o 115 120 125 Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val Tyr Lys 130 135 140 Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e Al a Lys 145 150 155 160 Pr o Ar g Pr o Pr o Tr p Met Gl y Leu Leu Gl y Pr o Thr I l e Gl n Al a Gl u 165 170 175 Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser Hi s Pr o 180 185 190 Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y 195 200 205 Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys

   Page 401

   2018203206 08 May 2018

   97047_1

   210 215 220

   Val 225 Phe Pr o Gl y Gl y Ser 230 Hi s Thr Tyr Val Tr p 235 Gl n Val Leu Lys Gl u 240 As n Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser Tyr Leu 245 250 255 Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e Gl y Al a 260 265 270 Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr Gl n Thr 275 280 285 Leu Hi s Lys Phe Ile Leu Leu Phe Al a Val Phe As p Gl u Gl y Lys Ser 290 295 300 Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a 305 310 315 320 Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr Val As n 325 330 335 Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val Tyr Tr p 340 345 350

   Page 402

   97047_1

   2018203206 08 May 2018

   Hi s Val I l e 355 Gl y Met Gl y Thr Thr 360 Pr o Gl u Val Hi s Ser 365 I l e Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u 370 375 380 I l e Ser Pr o I l e Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met As p Leu 385 390 395 400 Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y 405 410 415 Met Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu 420 425 430 Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Tyr As p As p As p Leu Thr 435 440 445 As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser 450 455 460 Phe I l e Gl n I l e Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val 465 470 475 480 Hi s Tyr I l e Al a Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o Leu Val

   Page 403

   2018203206 08 May 2018

   97047_1

   485 490 495

   Leu Al a Pr o As p 500 As p Ar g Ser Tyr Lys 505 Ser Gl n Tyr Leu As n As n 510 Gl y Pr o Gl n Ar g I l e Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met Al a Tyr 515 520 525 Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y 530 535 540 I l e Leu Gl y Pr o Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu Leu I l e 545 550 555 560 I l e Phe Lys As n Gl n Al a Ser Ar g Pr o Tyr As n Ile Tyr Pr o Hi s Gl y 565 570 575 I l e Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys Gl y Val 580 585 590 Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe Lys Tyr 595 600 605 Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g 610 615 620

   Page 404

   97047_1

   2018203206 08 May 2018

   Cy s 625 Leu Thr Ar g Ty r Ty r 630 Ser Ser Phe Val As n 635 Met Gl u Ar g As p Leu 640 Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cy s Ty r Lys Gl u Ser Val 645 650 655 As p Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val I l e Leu 660 665 670 Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Ty r Leu Thr Gl u As n I l e 675 680 685 Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u 690 695 700 Phe Gl n Al a Ser As n Ile Met Hi s Ser Ile As n Gl y Ty r Val Phe As p 705 710 715 720 Ser Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p Ty r I l e 725 730 735 Leu Ser I l e Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y 740 745 750 Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr Leu Phe

   Page 405

   2018203206 08 May 2018

   97047_1

   755 760 765

   Pr o Phe Ser 770 Gl y Gl u Thr Val 775 Phe Met Ser Met Gl u 780 As n Pr o Gl y Leu Tr p I l e Leu Gl y Cys Hi s As n Ser As p Phe Ar g As n Ar g Gl y Met Thr 785 790 795 800 Al a Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p Ty r Ty r 805 810 815 Gl u As p Ser Tyr Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys As n As n 820 825 830 Al a I l e Gl u Pr o Ar g Ser Phe Ser Gl n As n Pr o Pr o Val Leu Lys Ar g 835 840 845 Hi s Gl n Al a Gl u Ile Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u 850 855 860 I l e As p Tyr As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u As p Phe 865 870 875 880 As p I l e Tyr As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys 885 890 895

   Page 406

   2018203206 08 May 2018

   97047 1

   Lys Thr Ar g Hi s 900 Ty r Phe Ile Al a Al a 905 Val Gl u Ar g Leu Tr p 910 As p Ty r Gl y Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser Gl y 915 920 925 Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y 930 935 940 Ser Phe Thr Gl n Pr o Leu Ty r Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y 945 950 955 960 Leu Leu Gl y Pr o Ty r Ile Ar g Al a Gl u Val Gl u As p As n I l e Met Val 965 970 975 Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Ty r Ser Phe Ty r Ser Ser Leu 980 985 990 I l e Ser Ty r Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl j Pro Arg Lys Asn 995 1000 1005

   Phe Val Lys Pro Asn Gl u Thr Lys Thr Tyr Phe Trp Lys Val Gl n 1010 1015 1020

   Hi s Hi s Met Al a Pro Thr Lys Asp Gl u Phe Asp Cys Lys Al a Trp

   Page 407

   2018203206 08 May 2018

   97047_1

   1025 1030 1035

   Al a Tyr 1040 Phe Ser As p Val As p 1045 Leu Gl u Lys As p Val 1050 Hi s Ser Gl y Leu I l e Gl y Pr o Leu Leu Val Cys Hi s Thr As n Thr Leu As n Pr o 1055 1060 1065 Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe 1070 1075 1080 Thr I l e Phe As p Gl u Thr Lys Ser Tr p Tyr Phe Thr Gl u As n Met 1085 1090 1095 Gl u Ar g As n Cys Ar g Al a Pr o Cys As n Ile Gl n Met Gl u As p Pr o 1100 1105 1110 Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s Al a Ile As n Gl y Tyr I l e 1115 1120 1125 Met As p Thr Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n Ar g I l e 1130 1135 1140

   Ar g Tr p

   Tyr Leu Leu Ser Met

   Gl y Ser Asn Gl u Asn

   I l e Hi s Ser

   1145

   1150

   1155

   Page 408

   2018203206 08 May 2018

   97047_1

   I l e Hi s 1160 Phe Ser Gl y Hi s Val 1165 Phe Thr Val Ar g Lys 1170 Lys Gl u Gl u Tyr Lys Met Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe Gl u Thr 1175 1180 1185 Val Gl u Met Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys 1190 1195 1200 Leu I l e Gl y Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu 1205 1210 1215 Val Tyr Ser As n Lys Cys Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y 1220 1225 1230 Hi s I l e Ar g As p Phe Gl n Ile Thr Al a Ser Gl y Gl n Tyr Gl y Gl n 1235 1240 1245 Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser I l e As n 1250 1255 1260 Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu 1265 1270 1275 Leu Al a Pr o Met Ile Ile Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g

   Page 409

   2018203206 08 May 2018

   1280 1285 97047 _1 1290 Gl n Lys Phe Ser Ser Leu Tyr Ile Ser Gl n Phe Ile I l e Met Tyr 1295 1300 1305 Ser Leu As p Gl y Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr 1310 1315 1320 Gl y Thr Leu Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y I l e 1325 1330 1335 Lys Hi s As n Ile Phe As n Pr o Pr o Ile Ile Al a Ar g Tyr I l e Ar g 1340 1345 1350 Leu Hi s Pr o Thr Hi s Tyr Ser Ile Ar g Ser Thr Leu Ar g Met Gl u 1355 1360 1365 Leu Met Gl y Cys As p Leu As n Ser Cys Ser Met Pr o Leu Gl y Met 1370 1375 1380 Gl u Ser Lys Al a Ile Ser As p Al a Gl n Ile Thr Al a Ser Ser Tyr 1385 1390 1395 Phe Thr As n Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu 1400 1405 1410

   Page 410

   2018203206 08 May 2018

   97047 1

   Hi s Leu 1415 Gl n Gl y Ar g Ser As n 1420 Al a Tr p Ar g Pr o Gl n 1425 Val As n As n Pr o Lys Gl u Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val 1430 1435 1440 Thr Gl y Val Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met 1445 1450 1455 Ty r Val Lys Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n 1460 1465 1470 Tr p Thr Leu Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y 1475 1480 1485 As n Gl n As p Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o 1490 1495 1500 Leu Leu Thr Arg Ty r Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s 1505 1510 1515 Gl n I l e Al a Leu Ar g Met Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p 1520 1525 1530 Leu Ty r As p Lys Thr Hi s Thr Cy s Pr o Pr o Cy s Pr o Al a Pr o Gl u

   Page 411

   2018203206 08 May 2018

   1535 1540 97047 _1 1545 Leu Leu Gly Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys 1550 1555 1560 As p Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val 1565 1570 1575 Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Ty r 1580 1585 1590 Val As p Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u 1595 1600 1605 Gl u Gl n Tyr As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val 1610 1615 1620 Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cy s Lys Val 1625 1630 1635 Ser As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr I l e Ser Lys 1640 1645 1650 Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o 1655 1660 1665

   Page 412

   2018203206 08 May 2018

   97047_1

   Ser Ar g 1670 As p Gl u Leu Thr Lys 1675 As n Gl n Val Ser Leu 1680 Thr Cys Leu Val Lys Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser 1685 1690 1695 As n Gl y Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu 1700 1705 1710 As p Ser As p Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p 1715 1720 1725 Lys Ser Arg Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met 1730 1735 1740 Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu 1745 1750 1755

   Ser Pr o Gl y Lys 1760 <210> 116 <211> 1726 <212> PRT <213> Artificial Sequence

   Page 413

   2018203206 08 May 2018

   97047_1 <220>

   <223> pSYNFVI I I 271 protein sequence <400> 116

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cys Phe Phe 10 Leu Cys Leu Leu Ar g 15 Phe Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Tyr Met Gl n Gl y Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 35 40 45 Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 50 55 60 Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Al a Ser Ser Ser 65 70 75 80 As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g Phe Pr o Pr o Ar g Val Pr o 85 90 95 Lys Ser Phe Pr o Phe As n Thr Ser Val Val Tyr Lys Lys Thr Leu Phe 100 105 110 Val Gl u Phe Thr As p Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o

   Page 414

   2018203206 08 May 2018

   97047_1

   115 120 125

   Tr p Met Gl y Leu Leu Gl y Pr o 135 Thr Ile Gl n Al a Gl u 140 Val Tyr As p Thr 130 Val Val I l e Thr Leu Lys As n Met Al a Ser Hi s Pr o Val Ser Leu Hi s 145 150 155 160 Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a Gl u Tyr As p 165 170 175 As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p As p Lys Val Phe Pr o Gl y 180 185 190 Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met 195 200 205 Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser Tyr Leu Ser Hi s Val As p 210 215 220 Leu Val Lys As p Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cys 225 230 235 240

   Arg Gl u Gl y Ser

   Leu Al a Lys Gl u

   Lys Thr Gl n Thr

   Leu Hi s Ly s Phe

   245

   250

   255

   Page 415

   2018203206 08 May 2018

   97047_1

   I l e Leu Leu Phe 260 Al a Val Phe As p Gl u 265 Gl y Lys Ser Tr p Hi s 270 Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p Al a Al a Ser Al a Ar g Al a 275 280 285 Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr Val As n Ar g Ser Leu Pr o 290 295 300 Gl y Leu I l e Gl y Cys Hi s Ar g Lys Ser Val Tyr Tr p Hi s Val I l e Gl y 305 310 315 320 Met Gl y Thr Thr Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr 325 330 335 Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser Leu Gl u I l e Ser Pr o I l e 340 345 350 Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu 355 360 365 Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s As p Gl y Met Gl u Al a Tyr 370 375 380 Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n

   Page 416

   2018203206 08 May 2018

   97047_1

   385 390 395 400

   As n Gl u Gl u Al a Gl u 405 As p Tyr Asp Asp Asp Leu Thr 410 As p Ser Gl u 415 Met As p Val Val Ar g Phe As p As p As p As n Ser Pr o Ser Phe I l e Gl n I l e 420 425 430 Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Tyr I l e Al a 435 440 445 Al a Gl u Gl u Gl u As p Tr p As p Tyr Al a Pr o Leu Val Leu Al a Pr o As p 450 455 460 As p Ar g Ser Tyr Lys Ser Gl n Tyr Leu As n As n Gl y Pr o Gl n Ar g I l e 465 470 475 480 Gl y Ar g Lys Tyr Lys Lys Val Ar g Phe Met Al a Tyr Thr As p Gl u Thr 485 490 495 Phe Lys Thr Ar g Gl u Al a Ile Gl n Hi s Gl u Ser Gl y I l e Leu Gl y Pr o 500 505 510 Leu Leu Tyr Gl y Gl u Val Gl y As p Thr Leu Leu Ile I l e Phe Lys As n 515 520 525

   Page 417

   2018203206 08 May 2018

   97047 1

   Gl n Al a 530 Ser Ar g Pro Tyr As n 535 Ile Tyr Pr o Hi s Gl y 540 I l e Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys 545 550 555 560 As p Phe Pr o I l e Leu Pr o Gl y Gl u Ile Phe Lys Tyr Lys Tr p Thr Val 565 570 575 Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p Pr o Ar g Cys Leu Thr Ar g 580 585 590 Tyr Tyr Ser Ser Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu 595 600 605 I l e Gly Pr o Leu Leu Ile Cys Tyr Lys Gl u Ser Val As p Gl n Ar g Gl y 610 615 620 As n Gl n I l e Met Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe 625 630 635 640 As p Gl u As n Ar g Ser Tr p Tyr Leu Thr Gl u As n Ile Gl n Ar g Phe Leu 645 650 655 Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser

   Page 418

   2018203206 08 May 2018

   660 665 97047_1 670 As n I l e Met Hi s Ser Ile As n Gl y Ty r Val Phe As p Ser Leu Gl n Leu 675 680 685 Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p Ty r Ile Leu Ser I l e Gl y 690 695 700 Al a Gl n Thr As p Phe Leu Ser Val Phe Phe Ser Gl y Ty r Thr Phe Lys 705 710 715 720 Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y 725 730 735 Gl u Thr Val Phe Met Ser Met Gl u As n Pr o Gl y Leu Tr p I l e Leu Gl y 740 745 750 Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys 755 760 765 Val Ser Ser Cy s As p Lys As n Thr Gl y As p Ty r Ty r Gl u As p Ser Ty r 770 775 780 Gl u As p I l e Ser Al a Ty r Leu Leu Ser Lys As n As n Al a I l e Gl u Pr o 785 790 795 800

   Page 419

   2018203206 08 May 2018

   97047 1

   Ar g Ser Phe Ser Gl n As n 805 Pr o Pr o Val Leu 810 Lys Ar g Hi s Gl n Al a 815 Gl u I l e Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u I l e As p Tyr As p 820 825 830 As p Thr I l e Ser Val Gl u Met Lys Lys Gl u As p Phe As p I l e Tyr As p 835 840 845 Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys Thr Ar g Hi s 850 855 860 Tyr Phe I l e Al a Al a Val Gl u Ar g Leu Tr p As p Tyr Gl y Met Ser Ser 865 870 875 880 Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n Ser Gl y Ser Val Pr o Gl n 885 890 895 Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr As p Gl y Ser Phe Thr Gl n 900 905 910 Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s Leu Gl y Leu Leu Gl y Pr o 915 920 925 Tyr I l e Ar g Al a Gl u Val Gl u As p As n Ile Met Val Thr Phe Ar g As n

   Page 420

   2018203206 08 May 2018

   97047_1

   930 935 940

   Gl n 945 Al a Ser Ar g Pr o Tyr 950 Ser Phe Tyr Ser Ser 955 Leu I l e Ser Tyr Gl u 960 Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g Lys As n Phe Val Lys Pr o 965 970 975 As n Gl u Thr Lys Thr Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met Al a Pr o 980 985 990 Thr Lys As p Gl u Phe As p Cys Lys Al a Tr p Al a Ty r Phe Ser As p Val

   995 1000 1005 Asp Leu Gl u Lys Asp Val Hi s Ser Gl y Leu I l e Gl y Pr o Leu Leu 1010 1015 1020 Val Cys Hi s Thr Asn Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val 1025 1030 1035 Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe As p Gl u Thr 1040 1045 1050 Lys Ser Trp Tyr Phe Thr Gl u Asn Met Gl u Arg Asn Cys Arg Al a 1055 1060 1065

   Page 421

   2018203206 08 May 2018

   97047_1

   Pr o Cys As n Ile Gl n Met Gl u As p Pr o Thr Phe Lys Gl u As n Tyr 1070 1075 1080 Ar g Phe Hi s Al a Ile As n Gl y Tyr Ile Met As p Thr Leu Pr o Gl y 1085 1090 1095 Leu Val Met Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr Leu Leu Ser 1100 1105 1110 Met Gl y Ser As n Gl u As n Ile Hi s Ser Ile Hi s Phe Ser Gl y Hi s 1115 1120 1125 Val Phe Thr Val Ar g Lys Lys Gl u Gl u Tyr Lys Met Al a Leu Tyr 1130 1135 1140 As n Leu Tyr Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser 1145 1150 1155 Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys Leu Ile Gl y Gl u Hi s Leu 1160 1165 1170 Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Tyr Ser As n Lys Cys 1175 1180 1185 Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n

   Page 422

   2018203206 08 May 2018

   1190 1195 97047 _1 1200 I l e Thr Al a Ser Gl y Gl n Tyr Gl y Gl n Tr p Al a Pr o Lys Leu Al a 1205 1210 1215 Ar g Leu Hi s Tyr Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u 1220 1225 1230 Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o Met I l e I l e 1235 1240 1245 Hi s Gl y I l e Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu 1250 1255 1260 Tyr I l e Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p Gl y Lys Lys 1265 1270 1275 Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe 1280 1285 1290 Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n I l e Phe As n 1295 1300 1305 Pr o Pr o I l e Ile Al a Ar g Tyr Ile Ar g Leu Hi s Pr o Thr Hi s Tyr 1310 1315 1320

   Page 423

   2018203206 08 May 2018

   97047_1

   Ser I l e 1325 Ar g Ser Thr Leu Ar g 1330 Met Gl u Leu Met Gl y 1335 Cy s As p Leu As n Ser Cy s Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a I l e Ser 1340 1345 1350 As p Al a Gl n Ile Thr Al a Ser Ser Ty r Phe Thr As n Met Phe Al a 1355 1360 1365 Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n Gl y Ar g Ser 1370 1375 1380 As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u Tr p Leu Gl n 1385 1390 1395 Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val Thr Thr Gl n 1400 1405 1410 Gl y Val Lys Ser Leu Leu Thr Ser Met Ty r Val Lys Gl u Phe Leu 1415 1420 1425 I l e Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n 1430 1435 1440 As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr

   Page 424

   2018203206 08 May 2018

   1445 1450 97047 _1 1455 Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr Ar g Ty r Leu 1460 1465 1470 Ar g I l e Hi s Pr o Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met 1475 1480 1485 Gl u Val Leu Gl y Cy s Gl u Al a Gl n As p Leu Ty r As p Lys Thr Hi s 1490 1495 1500 Thr Cy s Pr o Pr o Cy s Pr o Al a Pr o Gl u Leu Leu Gl y Gl y Pr o Ser 1505 1510 1515 Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu Met I l e Ser 1520 1525 1530 Ar g Thr Pr o Gl u Val Thr Cy s Val Val Val As p Val Ser Hi s Gl u 1535 1540 1545 As p Pr o Gl u Val Lys Phe As n Tr p Ty r Val As p Gl y Val Gl u Val 1550 1555 1560 Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Ty r As n Ser Thr 1565 1570 1575

   Page 425

   2018203206 08 May 2018

   97047_1

   Ty r Ar g 1580 Val Val Ser Val Leu 1585 Thr Val Leu Hi s Gl n 1590 As p Tr p Leu As n Gl y Lys Gl u Ty r Lys Cy s Lys Val Ser As n Lys Al a Leu Pr o 1595 1600 1605 Al a Pr o I l e Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g 1610 1615 1620 Gl u Pr o Gl n Val Ty r Thr Leu Pr o Pr o Ser Ar g As p Gl u Leu Thr 1625 1630 1635 Lys As n Gl n Val Ser Leu Thr Cy s Leu Val Lys Gl y Phe Ty r Pr o 1640 1645 1650 Ser As p I l e Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n 1655 1660 1665 As n Ty r Lys Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe 1670 1675 1680 Phe Leu Ty r Ser Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n 1685 1690 1695 Gl y As n Val Phe Ser Cy s Ser Val Met Hi s Gl u Al a Leu Hi s As n

   Page 426

   2018203206 08 May 2018

   1700

   97047_1

   1705 1710

   His Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pro Gly Lys 1715 1720 1725

   <210> 117 <211> 1901 <212> PRT <213> Ar t i f i ci al Sequence <220> <223> pSYN FVI I I pr ot ei n sequence 272

   <400> 117

   Met Gl n I l e Gl u Leu Ser Thr Cys Phe Phe Le u Cys Leu Leu Ar g Ph e 1 5 10 15 Cys Phe Ser Al a Thr Ar g Ar g Tyr Tyr Leu Gl ^y Al a Val Gl u Leu Se r 20 25 30 Tr p As p Tyr Met Gl n Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u 35 40 45 Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl ^y Ser Gl u Thr Pr o Gl ^y 50 55 60 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a

   Page 427

   2018203206 08 May 2018

   97047_1

   65 70 75 80

   Thr Ser Gl y Ser Gl u 85 Thr Pr o Gl y Thr Ser 90 Gl u Ser Al a Thr Pr o 95 Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 100 105 110 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u Ser 115 120 125 Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser 130 135 140 Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 145 150 155 160 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y 165 170 175 Ser Pr o Thr Ser Thr Gl u Gl u Gl y Al a Ser Ser Ser As p Leu Gl y Gl u 180 185 190 Leu Pr o Val Asp Al a Ar g Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o 195 200 205

   Page 428

   97047_1

   2018203206 08 May 2018

   Phe As n 210 Thr Ser Val Val Tyr 215 Lys Lys Thr Leu Phe 220 Val Gl u Phe Thr As p Hi s Leu Phe As n Ile Al a Lys Pr o Ar g Pr o Pr o Tr p Met Gl y Leu 225 230 235 240 Leu Gl y Pr o Thr Ile Gl n Al a Gl u Val Tyr As p Thr Val Val I l e Thr 245 250 255 Leu Lys As n Met Al a Ser Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val 260 265 270 Ser Tyr Tr p Lys Al a Ser Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser 275 280 285 Gl n Ar g Gl u Lys Gl u As p As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr 290 295 300 Tyr Val Tr p Gl n Val Leu Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o 305 310 315 320 Leu Cys Leu Thr Tyr Ser Tyr Leu Ser Hi s Val As p Leu Val Lys As p 325 330 335 Leu As n Ser Gl y Leu Ile Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser

   Page 429

   2018203206 08 May 2018

   970 47_1 340 345 350 Leu Al a Lys Gl u Lys Thr Gl n Thr Leu Hi s Lys Phe I l e Leu Leu Phe 355 360 365 Al a Val Phe As p Gl u Gl y Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser 370 375 380 Leu Met Gl n As p Ar g As p Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met 385 390 395 400 Hi s Thr Val As n Gl y Ty r Val As n Ar g Ser Leu Pr o Gl y Leu I l e Gl y 405 410 415 Cy s Hi s Ar g Lys Ser Val Ty r Tr p Hi s Val Ile Gl y Met Gl y Thr Thr 420 425 430 Pr o Gl u Val Hi s Ser Ile Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g 435 440 445 Asn Hi s Ar g Gl n Al a Ser Leu Gl u Ile Ser Pr o Ile Thr Phe Leu Thr 450 455 460 Al a Gl n Thr Leu Leu Met As p Leu Gl y Gl n Phe Leu Leu Phe Cy s Hi s 465 470 475 480

   Page 430

   97047_1

   2018203206 08 May 2018

   I l e Ser Ser Hi s Gl n 485 Hi s As p Gl y Met Gl u 490 Al a Tyr Val Lys Val 495 As p Ser Cys Pr o Gl u Gl u Pr o Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a 500 505 510 Gl u As p Tyr As p As p As p Leu Thr As p Ser Gl u Met As p Val Val Ar g 515 520 525 Phe As p As p As p As n Ser Pr o Ser Phe Ile Gl n Ile Ar g Ser Val Al a 530 535 540 Lys Lys Hi s Pr o Lys Thr Tr p Val Hi s Tyr Ile Al a Al a Gl u Gl u Gl u 545 550 555 560 Asp Tr p As p Tyr Al a Pr o Leu Val Leu Al a Pr o As p As p Ar g Ser Tyr 565 570 575 Lys Ser Gl n Tyr Leu As n As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Tyr 580 585 590 Lys Lys Val Ar g Phe Met Al a Tyr Thr As p Gl u Thr Phe Lys Thr Ar g 595 600 605 Gl u Al a I l e Gl n Hi s Gl u Ser Gl y Ile Leu Gl y Pr o Leu Leu Tyr Gl y

   Page 431

   2018203206 08 May 2018

   97047_1

   610 615 620

   Gl u 625 Val Gl y As p Thr Leu 630 Leu Ile Ile Phe Lys 635 As n Gl n Al a Ser Ar g 640 Pr o Ty r As n I l e Ty r Pr o Hi s Gl y Ile Thr As p Val Ar g Pr o Leu Ty r 645 650 655 Ser Ar g Ar g Leu Pr o Lys Gl y Val Lys Hi s Leu Lys As p Phe Pr o I l e 660 665 670 Leu Pr o Gl y Gl u Ile Phe Lys Ty r Lys Tr p Thr Val Thr Val Gl u As p 675 680 685 Gl y Pr o Thr Lys Ser As p Pr o Ar g Cy s Leu Thr Ar g Ty r Ty r Ser Ser 690 695 700 Phe Val As n Met Gl u Ar g As p Leu Al a Ser Gl y Leu I l e Gl y Pr o Leu 705 710 715 720 Leu I l e Cy s Ty r Lys Gl u Ser Val As p Gl n Ar g Gl y As n Gl n I l e Met 725 730 735 Ser As p Lys Ar g As n Val Ile Leu Phe Ser Val Phe As p Gl u As n Ar g 740 745 750

   Page 432

   97047_1

   2018203206 08 May 2018

   Ser Tr p Tyr 755 Leu Thr Gl u As n I l e Gl n Ar g 760 Phe Leu Pr o 765 As n Pr o Al a Gl y Val Gl n Leu Gl u As p Pr o Gl u Phe Gl n Al a Ser As n I l e Met Hi s 770 775 780 Ser I l e As n Gl y Tyr Val Phe As p Ser Leu Gl n Leu Ser Val Cys Leu 785 790 795 800 Hi s Gl u Val Al a Tyr Tr p Tyr Ile Leu Ser Ile Gl y Al a Gl n Thr As p 805 810 815 Phe Leu Ser Val Phe Phe Ser Gl y Tyr Thr Phe Lys Hi s Lys Met Val 820 825 830 Tyr Gl u As p Thr Leu Thr Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe 835 840 845 Met Ser Met Gl u As n Pr o Gl y Leu Tr p Ile Leu Gl y Cys Hi s As n Ser 850 855 860 Asp Phe Ar g As n Ar g Gl y Met Thr Al a Leu Leu Lys Val Ser Ser Cys 865 870 875 880 As p Lys As n Thr Gl y As p Tyr Tyr Gl u As p Ser Tyr Gl u As p I l e Ser

   Page 433

   2018203206 08 May 2018

   97047_1

   885 890 895

   Al a Tyr Leu Leu Ser Ly s As n As n Al a Ile Gl u Pr o Ar g Se r Phe Ser 900 905 91 0 Gl n As n Gl y Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 915 920 925 Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl ^y Thr Ser 930 935 940 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser 945 95 0 955 960 Gl y Ser Gl u Thr Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y 965 970 975 Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl ^y Ser Pr o 980 985 99 0

   Al a Gl y Ser

   Pr o Thr Ser Thr

   Gl u

   Gl u Gl y Thr Ser Gl u Ser Al a Thr

   995

   1000

   1005

   Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u

   1010 1015 1020

   Page 434

   97047_1

   2018203206 08 May 2018

   Thr Pr o 1025 Gl y Thr Ser Gl u Ser 1030 Al a Thr Pr o Gl u Ser 1035 Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a 1040 1045 1050 Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser 1055 1060 1065 Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser 1070 1075 1080 Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 1085 1090 1095 Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 1100 1105 1110 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser 1115 1120 1125 Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr 1130 1135 1140 Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y

   Page 435

   2018203206 08 May 2018

   1145 1150 97047 _1 1155 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o 1160 1165 1170 Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr 1175 1180 1185 Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser 1190 1195 1200 Al a Pr o Al a Ser Ser Pr o Pr o Val Leu Lys Ar g Hi s Gl n Al a Gl u 1205 1210 1215 I l e Thr Ar g Thr Thr Leu Gl n Ser As p Gl n Gl u Gl u I l e As p Tyr 1220 1225 1230 As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u As p Phe As p I l e 1235 1240 1245 Tyr As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe Gl n Lys Lys 1250 1255 1260 Thr Ar g Hi s Tyr Phe Ile Al a Al a Val Gl u Ar g Leu Tr p As p Tyr 1265 1270 1275

   Page 436

   97047_1

   2018203206 08 May 2018

   Gly Met 1280 Ser Ser Ser Pr o Hi s 1285 Val Leu Ar g As n Ar g 1290 Al a Gl n Ser Gly Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr 1295 1300 1305 Asp Gl y Ser Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u 1310 1315 1320 Hi s Leu Gl y Leu Leu Gl y Pr o Tyr Ile Ar g Al a Gl u Val Gl u As p 1325 1330 1335 Asn I l e Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser 1340 1345 1350 Phe Tyr Ser Ser Leu Ile Ser Tyr Gl u Gl u As p Gl n Ar g Gl n Gl y 1355 1360 1365 Al a Gl u Pr o Ar g Lys As n Phe Val Lys Pr o As n Gl u Thr Lys Thr 1370 1375 1380 Tyr Phe Tr p Lys Val Gl n Hi s Hi s Met Al a Pr o Thr Lys As p Gl u 1385 1390 1395 Phe As p Cys Lys Al a Tr p Al a Tyr Phe Ser As p Val As p Leu Gl u

   Page 437

   2018203206 08 May 2018

   97047_1

   1400 1405 1410

   Lys As p 1415 Val Hi s Ser Gl y Leu 1420 Ile Gl y Pr o Leu Leu 1425 Val Cys Hi s Thr As n Thr Leu As n Pr o Al a Hi s Gl y Ar g Gl n Val Thr Val Gl n 1430 1435 1440 Gl u Phe Al a Leu Phe Phe Thr Ile Phe As p Gl u Thr Lys Ser Tr p 1445 1450 1455 Tyr Phe Thr Gl u As n Met Gl u Ar g As n Cys Ar g Al a Pr o Cys As n 1460 1465 1470 I l e Gl n Met Gl u As p Pr o Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s 1475 1480 1485 Al a I l e As n Gl y Tyr Ile Met As p Thr Leu Pr o Gl y Leu Val Met 1490 1495 1500 Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr Leu Leu Ser Met Gl y Ser 1505 1510 1515 As n Gl u As n Ile Hi s Ser Ile Hi s Phe Ser Gl y Hi s Val Phe Thr

   1520 1525 1530

   Page 438

   97047_1

   2018203206 08 May 2018

   Val Ar g 1535 Lys Lys Gl u Gl u Ty r 1540 Lys Met Al a Leu Ty r 1545 As n Leu Ty r Pr o Gl y Val Phe Gl u Thr Val Gl u Met Leu Pr o Ser Lys Al a Gl y 1550 1555 1560 I l e Tr p Ar g Val Gl u Cy s Leu Ile Gl y Gl u Hi s Leu Hi s Al a Gl y 1565 1570 1575 Met Ser Thr Leu Phe Leu Val Ty r Ser As n Lys Cy s Gl n Thr Pr o 1580 1585 1590 Leu Gl y Met Al a Ser Gl y Hi s Ile Ar g As p Phe Gl n I l e Thr Al a 1595 1600 1605 Ser Gl y Gl n Ty r Gl y Gl n Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s 1610 1615 1620 Ty r Ser Gl y Ser Ile As n Al a Tr p Ser Thr Lys Gl u Pr o Phe Ser 1625 1630 1635 Tr p I l e Lys Val As p Leu Leu Al a Pr o Met Ile Ile Hi s Gl y I l e 1640 1645 1650 Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe Ser Ser Leu Ty r I l e Ser

   Page 439

   2018203206 08 May 2018

   1655 1660 97047 _1 1665 Gl n Phe I l e Ile Met Tyr Ser Leu As p Gl y Lys Lys Tr p Gl n Thr 1670 1675 1680 Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu Met Val Phe Phe Gl y As n 1685 1690 1695 Val As p Ser Ser Gl y Ile Lys Hi s As n Ile Phe As n Pr o Pr o I l e 1700 1705 1710 I l e Al a Ar g Tyr Ile Ar g Leu Hi s Pr o Thr Hi s Tyr Ser I l e Ar g 1715 1720 1725 Ser Thr Leu Ar g Met Gl u Leu Met Gl y Cys As p Leu As n Ser Cys 1730 1735 1740 Ser Met Pr o Leu Gl y Met Gl u Ser Lys Al a Ile Ser As p Al a Gl n 1745 1750 1755 I l e Thr Al a Ser Ser Tyr Phe Thr As n Met Phe Al a Thr Tr p Ser 1760 1765 1770 Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n Gl y Ar g Ser As n Al a Tr p 1775 1780 1785

   Page 440

   97047_1

   2018203206 08 May 2018

   Ar g Pr o 1790 Gl n Val As n As n Pr o 1795 Lys Gl u Tr p Leu Gl n 1800 Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val Thr Thr Gl n Gl y Val Lys 1805 1810 1815 Ser Leu Leu Thr Ser Met Tyr Val Lys Gl u Phe Leu I l e Ser Ser 1820 1825 1830 Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu Phe Phe Gl n As n Gl y Lys 1835 1840 1845 Val Lys Val Phe Gl n Gl y As n Gl n As p Ser Phe Thr Pr o Val Val 1850 1855 1860 As n Ser Leu As p Pr o Pr o Leu Leu Thr Ar g Tyr Leu Ar g I l e Hi s 1865 1870 1875 Pr o Gl n Ser Tr p Val Hi s Gl n Ile Al a Leu Ar g Met Gl u Val Leu 1880 1885 1890

   Gl y Cys

   Gl u Al a Gl n Asp Leu

   Tyr

   1895

   1900 <210> 118

   Page 441

   2018203206 08 May 2018

   97047_1 <211> 1515 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN VWF 031 pr ot ei n sequence <400> 118

   Met 1 I l e Pr o Al a Ar g 5 Phe Al a Gl y Val Leu 10 Leu Al a Leu Al a Leu 15 I l e Leu Pr o Gl y Thr Leu Cy s Al a Gl u Gl y Thr Ar g Gl y Ar g Ser Ser Thr 20 25 30 Al a Ar g Cy s Ser Leu Phe Gl y Ser As p Phe Val As n Thr Phe As p Gl y 35 40 45 Ser Met Ty r Ser Phe Al a Gl y Ty r Cy s Ser Ty r Leu Leu Al a Gl y Gl y 50 55 60 Cy s Gl n Lys Ar g Ser Phe Ser Ile Ile Gl y As p Phe Gl n As n Gl y Lys 65 70 75 80 Ar g Val Ser Leu Ser Val Ty r Leu Gl y Gl u Phe Phe As p I l e Hi s Leu 85 90 95 Phe Val As n Gl y Thr Val Thr Gl n Gl y As p Gl n Ar g Val Ser Met Pr o

   Page 442

   2018203206 08 May 2018

   100 105 97047_1 110 Tyr Al a Ser Lys Gl y Leu Tyr Leu Gl u Thr Gl u Al a Gl y Tyr Tyr Lys 115 120 125 Leu Ser Gl y Gl u Al a Tyr Gl y Phe Val Al a Ar g Ile As p Gl y Ser Gl y 130 135 140 As n Phe Gl n Val Leu Leu Ser As p Ar g Tyr Phe As n Lys Thr Cys Gl y 145 150 155 160 Leu Cys Gl y As n Phe As n Ile Phe Al a Gl u As p As p Phe Met Thr Gl n 165 170 175 Gl u Gl y Thr Leu Thr Ser As p Pr o Tyr As p Phe Al a As n Ser Tr p Al a 180 185 190 Leu Ser Ser Gl y Gl u Gl n Tr p Cys Gl u Ar g Al a Ser Pr o Pr o Ser Ser 195 200 205 Ser Cys As n I l e Ser Ser Gl y Gl u Met Gl n Lys Gl y Leu Tr p Gl u Gl n 210 215 220 Cys Gl n Leu Leu Lys Ser Thr Ser Val Phe Al a Ar g Cys Hi s Pr o Leu 225 230 235 240

   Page 443

   97047_1

   2018203206 08 May 2018

   Val As p Pr o Gl u Pr o 245 Phe Val Al a Leu Cy s 250 Gl u Lys Thr Leu Cy s 255 Gl u Cy s Al a Gl y Gl y Leu Gl u Cy s Al a Cy s Pr o Al a Leu Leu Gl u Ty r Al a 260 265 270 Ar g Thr Cy s Al a Gl n Gl u Gl y Met Val Leu Ty r Gl y Tr p Thr As p Hi s 275 280 285 Ser Al a Cy s Ser Pr o Val Cy s Pr o Al a Gl y Met Gl u Ty r Ar g Gl n Cy s 290 295 300 Val Ser Pr o Cy s Al a Ar g Thr Cy s Gl n Ser Leu Hi s I l e As n Gl u Met 305 310 315 320 Cy s Gl n Gl u Ar g Cy s Val As p Gl y Cy s Ser Cy s Pr o Gl u Gl y Gl n Leu 325 330 335 Leu As p Gl u Gl y Leu Cy s Val Gl u Ser Thr Gl u Cy s Pr o Cy s Val Hi s 340 345 350 Ser Gl y Lys Ar g Ty r Pr o Pr o Gl y Thr Ser Leu Ser Ar g As p Cy s As n 355 360 365 Thr Cy s I l e Cy s Ar g As n Ser Gl n Tr p Ile Cy s Ser As n Gl u Gl u Cy s

   Page 444

   2018203206 08 May 2018

   97047_1

   370 375 380

   Pr o 385 Gl y Gl u Cy s Leu Val 390 Thr Gl y Gl n Ser Hi s 395 Phe Lys Ser Phe As p 400 As n Ar g Ty r Phe Thr Phe Ser Gl y Ile Cy s Gl n Ty r Leu Leu Al a Ar g 405 410 415 As p Cy s Gl n As p Hi s Ser Phe Ser Ile Val Ile Gl u Thr Val Gl n Cy s 420 425 430 Al a As p As p Ar g As p Al a Val Cy s Thr Ar g Ser Val Thr Val Ar g Leu 435 440 445 Pr o Gl y Leu Hi s As n Ser Leu Val Lys Leu Lys Hi s Gl y Al a Gl y Val 450 455 460 Al a Met As p Gl y Gl n As p Ile Gl n Leu Pr o Leu Leu Lys Gl y As p Leu 465 470 475 480 Ar g I l e Gl n Hi s Thr Val Thr Al a Ser Val Ar g Leu Ser Ty r Gl y Gl u 485 490 495 As p Leu Gl n Met As p Tr p As p Gl y Ar g Gl y Ar g Leu Leu Val Lys Leu 500 505 510

   Page 445

   97047_1

   2018203206 08 May 2018

   Ser Pr o Val 515 Tyr Al a Gl y Lys Thr 520 Cys Gl y Leu Cys Gl y 525 As n Tyr As n Gl y As n Gl n Gl y As p As p Phe Leu Thr Pr o Ser Gl y Leu Al a Gl u Pr o 530 535 540 Arg Val Gl u As p Phe Gl y As n Al a Tr p Lys Leu Hi s Gl y As p Cys Gl n 545 550 555 560 As p Leu Gl n Lys Gl n Hi s Ser As p Pr o Cys Al a Leu As n Pr o Ar g Met 565 570 575 Thr Ar g Phe Ser Gl u Gl u Al a Cys Al a Val Leu Thr Ser Pr o Thr Phe 580 585 590 Gl u Al a Cys Hi s Ar g Al a Val Ser Pr o Leu Pr o Tyr Leu Ar g As n Cys 595 600 605 Ar g Tyr As p Val Cys Ser Cys Ser As p Gl y Ar g Gl u Cys Leu Cys Gl y 610 615 620 Al a Leu Al a Ser Tyr Al a Al a Al a Cys Al a Gl y Ar g Gl y Val Ar g Val 625 630 635 640 Al a Tr p Ar g Gl u Pr o Gl y Ar g Cys Gl u Leu As n Cys Pr o Lys Gl y Gl n

   Page 446

   2018203206 08 May 2018

   97047_1

   645 650 655

   Val Tyr Leu Gl n 660 Cys Gl y Thr Pro Cys Asn 665 Leu Thr Cys Ar g 670 Ser Leu Ser Tyr Pr o As p Gl u Gl u Cys As n Gl u Al a Cys Leu Gl u Gl y Cys Phe 675 680 685 Cys Pr o Pr o Gl y Leu Tyr Met As p Gl u Ar g Gl y As p Cys Val Pr o Lys 690 695 700 Al a Gl n Cys Pr o Cys Tyr Tyr As p Gl y Gl u Ile Phe Gl n Pr o Gl u As p 705 710 715 720 I l e Phe Ser As p Hi s Hi s Thr Met Cys Tyr Cys Gl u As p Gl y Phe Met 725 730 735 Hi s Cys Thr Met Ser Gl y Val Pr o Gl y Ser Leu Leu Pr o As p Al a Val 740 745 750 Leu Ser Ser Pr o Leu Ser Hi s Ar g Ser Lys Ar g Ser Leu Ser Cys Ar g 755 760 765 Pr o Pr o Met Val Lys Leu Val Cys Pr o Al a As p As n Leu Ar g Al a Gl u 770 775 780

   Page 447

   97047 1

   2018203206 08 May 2018

   Gl y 785 Leu Gl u Cy s Thr Lys 790 Thr Cys Gl n As n Tyr 795 As p Leu Gl u Cys Met 800 Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o Pr o Gl y Met Val Ar g 805 810 815 Hi s Gl u As n Ar g Cys Val Al a Leu Gl u Ar g Cys Pr o Cys Phe Hi s Gl n 820 825 830 Gl y Lys Gl u Tyr Al a Pr o Gl y Gl u Thr Val Lys Ile Gl y Cys As n Thr 835 840 845 Cys Val Cys Ar g As p Ar g Lys Tr p As n Cys Thr As p Hi s Val Cys As p 850 855 860 Al a Thr Cys Ser Thr Ile Gl y Met Al a Hi s Tyr Leu Thr Phe As p Gl y 865 870 875 880 Leu Lys Tyr Leu Phe Pr o Gl y Gl u Cys Gl n Tyr Val Leu Val Gl n As p 885 890 895 Tyr Cys Gl y Ser As n Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys 900 905 910 Gl y Cys Ser Hi s Pr o Ser Val Lys Cys Lys Lys Ar g Val Thr I l e Leu

   Page 448

   97047_1

   2018203206 08 May 2018

   915

   920

   925

   Val Gl u Gl y Gl y Gl u I l e Gl u Leu Phe Asp Gl y Gl u Val Asn Val Lys 930 935 940

   Ar g Pr o Met Lys Asp Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g 945 950 955 960

   Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975

   Hi s Leu Ser I l e Ser Val Val Leu Lys Gl n Thr Tyr Gl n Gl u Lys Val 980 985 990

   Cys Gl y Leu Cys Gl y Asn Phe Asp Gl y Ile Gl n Asn Asn Asp Leu Thr 995 1000 1005

   Ser Ser Asn Leu Gl n Val Gl u Gl u Asp Pro Val Asp Phe Gl y Asn 1010 1015 1020

   Ser Trp Lys Val Ser Ser Gl n Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035

   Leu Asp Ser Ser Pro Al a Thr Cys Hi s Asn Asn I l e Met Lys Gl n

   1040 1045 1050

   Page 449

   97047_1

   2018203206 08 May 2018

   Thr Met 1055 Val As p Ser Ser Cy s 1060 Ar g Ile Leu Thr Ser 1065 As p Val Phe Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu As p Val 1070 1075 1080 Cy s I l e Ty r As p Thr Cy s Ser Cy s Gl u Ser Ile Gl y As p Cy s Al a 1085 1090 1095 Al a Phe Cy s As p Thr Ile Al a Al a Ty r Al a Hi s Val Cy s Al a Gl n 1100 1105 1110 Hi s Gl y Lys Val Val Thr Tr p Ar g Thr Al a Thr Leu Cy s Pr o Gl n 1115 1120 1125 Ser Cy s Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Ty r Gl u Al a Gl u 1130 1135 1140 Tr p Ar g Ty r As n Ser Cy s Al a Pr o Al a Cy s Gl n Val Thr Cy s Gl n 1145 1150 1155 Hi s Pr o Gl u Pr o Leu Al a Cy s Pr o Val Gl n Cy s Val Gl u Gl y Cy s 1160 1165 1170 Hi s Al a Hi s Cy s Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu Leu Gl n

   Page 450

   97047_1

   2018203206 08 May 2018

   1175

   1180

   1185

   Thr Cys Val Asp Pro Gl u Asp Cys Pro Val Cys Gl u Val Al a Gl y 1190 1195 1200

   Arg Arg Phe Ala Ser Gly Lys Lys Val Thr Leu Asn Pro Ser Asp 1205 1210 1215

   Pro Gl u Hi s Cys Gl n Ile Cys Hi s Cys Asp Val Val Asn Leu Thr 1220 1225 1230

   Cys Gl u Ala Cys Gl n Gl u Pro Ile Ser Gly Gly Gly Gly Ser Gly 1235 1240 1245

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1250 1255 1260

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Leu Val Pr o Ar g Gl y Ser 1265 1270 1275

   Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Asp Lys Thr Hi s Thr 1280 1285 1290

   Cys Pro Pro Cys Pro Al a Pro Gl u Leu Leu Gly Gly Pro Ser Val

   1295 1300 1305

   Page 451

   97047_1

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   Phe Leu 1310 Phe Pr o Pr o Lys Pr o 1315 Lys As p Thr Leu Met 1320 I l e Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val Ser Hi s Gl u As p 1325 1330 1335 Pr o Gl u Val Lys Phe As n Tr p Tyr Val As p Gl y Val Gl u Val Hi s 1340 1345 1350 As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr As n Ser Thr Tyr 1355 1360 1365 Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n As p Tr p Leu As n 1370 1375 1380 Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys Al a Leu Pr o Al a 1385 1390 1395 Pr o I l e Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g Gl u 1400 1405 1410 Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p Gl u Leu Thr Lys 1415 1420 1425 As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y Phe Tyr Pr o Ser

   Page 452

   97047_1

   2018203206 08 May 2018

   As p 1430 I l e 1445 Al a 1435 Gl n 1440 Val Gl u Tr p Gl u 1450 Ser As n Gl y Pr o 1455 Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe Phe 1460 1465 1470 Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y 1475 1480 1485 As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s 1490 1495 1500 Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o Gl y Lys 1505 1510 1515

   <210> 119 <211> 1778 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN VWF 034 pr ot ei n sequence <400> 119

   Met I l e Pr o Al a Ar g Phe Al a Gl y Val Leu Leu Al a Leu Al a Leu I l e

   Page 453

   2018203206 08 May 2018

   97047_1

   1 5 10 15

   Leu Pr o Gl y Thr 20 Leu Cys Al a Gl u Gl y Thr 25 Ar g Gl y Ar g Ser 30 Ser Thr Al a Ar g Cys Ser Leu Phe Gl y Ser As p Phe Val As n Thr Phe As p Gl y 35 40 45 Ser Met Tyr Ser Phe Al a Gl y Tyr Cys Ser Tyr Leu Leu Al a Gl y Gl y 50 55 60 Cys Gl n Lys Ar g Ser Phe Ser Ile Ile Gl y As p Phe Gl n As n Gl y Lys 65 70 75 80 Ar g Val Ser Leu Ser Val Tyr Leu Gl y Gl u Phe Phe As p I l e Hi s Leu 85 90 95 Phe Val As n Gl y Thr Val Thr Gl n Gl y As p Gl n Ar g Val Ser Met Pr o 100 105 110 Tyr Al a Ser Lys Gl y Leu Tyr Leu Gl u Thr Gl u Al a Gl y Tyr Tyr Lys 115 120 125 Leu Ser Gl y Gl u Al a Tyr Gl y Phe Val Al a Ar g Ile As p Gl y Ser Gl y 130 135 140

   Page 454

   97047_1

   2018203206 08 May 2018

   As n 145 Phe Gl n Val Leu Leu 150 Ser As p Ar g Tyr Phe 155 As n Lys Thr Cys Gl y 160 Leu Cys Gl y As n Phe As n Ile Phe Al a Gl u As p As p Phe Met Thr Gl n 165 170 175 Gl u Gl y Thr Leu Thr Ser As p Pr o Tyr As p Phe Al a As n Ser Tr p Al a 180 185 190 Leu Ser Ser Gl y Gl u Gl n Tr p Cys Gl u Ar g Al a Ser Pr o Pr o Ser Ser 195 200 205 Ser Cys As n I l e Ser Ser Gl y Gl u Met Gl n Lys Gl y Leu Tr p Gl u Gl n 210 215 220 Cys Gl n Leu Leu Lys Ser Thr Ser Val Phe Al a Ar g Cys Hi s Pr o Leu 225 230 235 240 Val As p Pr o Gl u Pr o Phe Val Al a Leu Cys Gl u Lys Thr Leu Cys Gl u 245 250 255 Cys Al a Gl y Gl y Leu Gl u Cys Al a Cys Pr o Al a Leu Leu Gl u Tyr Al a 260 265 270 Ar g Thr Cys Al a Gl n Gl u Gl y Met Val Leu Tyr Gl y Tr p Thr As p Hi s

   Page 455

   2018203206 08 May 2018

   97047_1

   275 280 285

   Ser Al a Cys Ser Pr o Val Cys Pr o Al a Gl y Met Gl u Tyr Ar g Gl n Cys 290 295 300 Val Ser Pr o Cys Al a Ar g Thr Cy s Gl n Ser Leu Hi s I l e As n Gl u Met 305 310 315 320 Cys Gl n Gl u Ar g Cys Val As p Gl ^y Cys Ser Cys Pr o Gl u Gl y Gl n Leu 325 330 335 Leu As p Gl u Gl y Leu Cys Val Gl u Ser Thr Gl u Cys Pr o Cys Val Hi s 340 345 350 Ser Gl y Lys Ar g Tyr Pr o Pr o Gl ^y Thr Ser Leu Ser Ar g As p Cys As n 355 36 0 365 Thr Cys I l e Cys Ar g As n Ser Gl n Tr p Ile Cys Ser As n Gl u Gl u Cys 370 375 380 Pr o Gl y Gl u Cys Leu Val Thr Gl ^y Gl n Ser Hi s Phe Lys Ser Phe As p 385 390 395 400 As n Ar g Tyr Phe Thr Phe Ser Gl ^y Ile Cys Gl n Tyr Leu Leu Al a Ar g 405 410 415

   Page 456

   97047_1

   2018203206 08 May 2018

   As p Cy s Gl n As p 420 Hi s Ser Phe Ser Ile 425 Val Ile Gl u Thr Val 430 Gl n Cy s Al a As p As p Ar g As p Al a Val Cy s Thr Ar g Ser Val Thr Val Ar g Leu 435 440 445 Pr o Gl y Leu Hi s As n Ser Leu Val Lys Leu Lys Hi s Gl y Al a Gl y Val 450 455 460 Al a Met As p Gl y Gl n As p Ile Gl n Leu Pr o Leu Leu Lys Gl y As p Leu 465 470 475 480 Ar g I l e Gl n Hi s Thr Val Thr Al a Ser Val Ar g Leu Ser Ty r Gl y Gl u 485 490 495 As p Leu Gl n Met As p Tr p As p Gl y Ar g Gl y Ar g Leu Leu Val Lys Leu 500 505 510 Ser Pr o Val Ty r Al a Gl y Lys Thr Cy s Gl y Leu Cy s Gl y As n Ty r As n 515 520 525 Gl y As n Gl n Gl y As p As p Phe Leu Thr Pr o Ser Gl y Leu Al a Gl u Pr o 530 535 540 Ar g Val Gl u As p Phe Gl y As n Al a Tr p Lys Leu Hi s Gl y As p Cy s Gl n

   Page 457

   2018203206 08 May 2018

   97047_1

   545 550 555 560

   As p Leu Gl n Lys Gl n Hi s Ser As p Pr o Cys Al a Leu As n Pr o Ar g Met 565 570 575 Thr Ar g Phe Ser Gl u Gl u Al a Cys Al a Val Leu Thr Ser Pr o Thr Phe 580 585 590 Gl u Al a Cys Hi s Ar g Al a Val Ser Pr o Leu Pr o Tyr Leu Ar g As n Cys 595 600 605 Ar g Tyr As p Val Cys Se r Cys Ser As p Gl y Ar g Gl u Cys Leu Cys Gl y 610 615 620 Al a Leu Al a Ser Tyr Al a Al a Al a Cys Al a Gl y Ar g Gl y Val Ar g Val 625 63 0 635 640 Al a Tr p Ar g Gl u Pr o Gl ^y Ar g Cys Gl u Leu As n Cys Pr o Lys Gl y Gl n 645 650 655 Val Tyr Leu Gl n Cys Gl ^y Thr Pr o Cys As n Leu Thr Cys Ar g Ser Leu 660 665 670 Ser Tyr Pr o As p Gl u Gl u Cys As n Gl u Al a Cys Leu Gl u Gl y Cys Phe 675 680 685

   Page 458

   97047_1

   2018203206 08 May 2018

   Cys Pr o Pr o 690 Gl y Leu Tyr Met 695 As p Gl u Ar g Gl y Asp Cys 700 Val Pr o Lys Al a Gl n Cys Pr o Cys Tyr Tyr As p Gl y Gl u Ile Phe Gl n Pr o Gl u As p 705 710 715 720 I l e Phe Ser As p Hi s Hi s Thr Met Cys Tyr Cys Gl u As p Gl y Phe Met 725 730 735 Hi s Cys Thr Met Ser Gl y Val Pr o Gl y Ser Leu Leu Pr o As p Al a Val 740 745 750 Leu Ser Ser Pr o Leu Ser Hi s Ar g Ser Lys Ar g Ser Leu Ser Cys Ar g 755 760 765 Pr o Pr o Met Val Lys Leu Val Cys Pr o Al a As p As n Leu Ar g Al a Gl u 770 775 780 Gl y Leu Gl u Cys Thr Lys Thr Cys Gl n As n Tyr As p Leu Gl u Cys Met 785 790 795 800 Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o Pr o Gl y Met Val Ar g 805 810 815 Hi s Gl u As n Ar g Cys Val Al a Leu Gl u Ar g Cys Pr o Cys Phe Hi s Gl n

   Page 459

   2018203206 08 May 2018

   820 825 97047_1 830 Gl y Lys Gl u Ty r Al a Pr o Gl y Gl u Thr Val Lys Ile Gl y Cy s As n Thr 835 840 845 Cy s Val Cy s Ar g As p Ar g Lys Tr p As n Cy s Thr As p Hi s Val Cy s As p 850 855 860 Al a Thr Cy s Ser Thr Ile Gl y Met Al a Hi s Ty r Leu Thr Phe As p Gl y 865 870 875 880 Leu Lys Ty r Leu Phe Pr o Gl y Gl u Cy s Gl n Ty r Val Leu Val Gl n As p 885 890 895 Ty r Cy s Gl y Ser As n Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys 900 905 910 Gl y Cy s Ser Hi s Pr o Ser Val Lys Cy s Lys Lys Ar g Val Thr I l e Leu 915 920 925 Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y Gl u Val As n Val Lys 930 935 940 Arg Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g 945 950 955 960

   Page 460

   2018203206 08 May 2018

   97047_1

   Tyr Ile Ile Leu Leu Leu Gly Lys Ala Leu Ser Val Val Trp Asp Arg 965 970 975

   Hi s Leu Ser I l e Ser Val Val Leu Lys Gl n Thr Tyr Gl n Gl u Lys Val 980 985 990

   Cys Gl y Leu Cys Gl y Asn Phe Asp Gl y Ile Gl n Asn Asn Asp Leu Thr 995 1000 1005

   Ser Ser Asn Leu Gl n Val Gl u Gl u Asp Pro Val Asp Phe Gl y Asn 1010 1015 1020

   Ser Trp Lys Val Ser Ser Gl n Cys Ala Asp Thr Arg Lys Val Pro 1025 1030 1035

   Leu Asp Ser Ser Pro Al a Thr Cys Hi s Asn Asn I l e Met Lys Gl n 1040 1045 1050

   Thr Met Val Asp Ser Ser Cys Arg I l e Leu Thr Ser Asp Val Phe 1055 1060 1065

   Gl n Asp Cys Asn Lys Leu Val Asp Pro Gl u Pro Tyr Leu Asp Val 1070 1075 1080

   Cys I l e Tyr Asp Thr Cys Ser Cys Gl u Ser I l e Gl y Asp Cys Al a

   Page 461

   2018203206 08 May 2018

   97047_1

   1085 1090 1095

   Al a Phe 1100 Cy s As p Thr I l e Al a 1105 Al a Ty r Al a Hi s Val 1110 Cy s Al a Gl n Hi s Gl y Lys Val Val Thr Tr p Ar g Thr Al a Thr Leu Cy s Pr o Gl n 1115 1120 1125 Ser Cy s Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Ty r Gl u Al a Gl u 1130 1135 1140 Tr p Ar g Ty r Asn Ser Cy s Al a Pr o Al a Cy s Gl n Val Thr Cy s Gl n 1145 1150 1155 Hi s Pr o Gl u Pr o Leu Al a Cy s Pr o Val Gl n Cy s Val Gl u Gl y Cy s 1160 1165 1170 Hi s Al a Hi s Cy s Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu Leu Gl n 1175 1180 1185 Thr Cy s Val As p Pr o Gl u As p Cy s Pr o Val Cy s Gl u Val Al a Gl y 1190 1195 1200 Ar g Ar g Phe Al a Ser Gl y Lys Lys Val Thr Leu As n Pr o Ser As p

   1205 1210 1215

   Page 462

   2018203206 08 May 2018

   Pr o

   Cys

   Thr

   Gl u

   Gl y

   Gl u

   Ser

   Thr

   97047_1

   Gl u Hi s Cys Gl n Ile Cys Hi s Cys Asp Val Val Asn Leu Thr 1220 1225 1230

   Gl u Al a Cys Gl n Gl u Pro Ile Ser Gl y Thr Ser Gl u Ser Al a 1235 1240 1245

   Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser 1250 1255 1260

   Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 1265 1270 1275

   Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser 1280 1285 1290

   Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Thr Gl u Pr o 1295 1300 1305

   Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 1310 1315 1320

   Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 1325 1330 1335

   Gl y

   Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser

   Page 463

   2018203206 08 May 2018

   97047_1

   1340 1345 1350

   Gl u Ser 1355 Al a Thr Pr o Gl u Ser 1360 Gl y Pr o Gl y Ser Pr o 1365 Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 1370 1375 1380 Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 1385 1390 1395 Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser 1400 1405 1410 Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a 1415 1420 1425 Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser 1430 1435 1440 Gl u Thr Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 1445 1450 1455 Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser

   1460 1465 1470

   Page 464

   97047 1

   2018203206 08 May 2018

   Thr Gl u 1475 Pr o Ser Gl u Gl y Ser 1480 Al a Pr o Gl y Thr Ser 1485 Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser 1490 1495 1500 Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 1505 1510 1515 Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o As p I l e Gl y 1520 1525 1530 Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Leu Val Pr o Ar g Gl y 1535 1540 1545 Ser Gl y Gl y As p Lys Thr Hi s Thr Cy s Pr o Pr o Cy s Pr o Al a Pr o 1550 1555 1560 Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o 1565 1570 1575 Lys As p Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cy s Val 1580 1585 1590 Val Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p

   Page 465

   2018203206 08 May 2018

   1595 1600 97047 _1 1605 Tyr Val As p Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g 1610 1615 1620 Gl u Gl u Gl n Tyr As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr 1625 1630 1635 Val Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys 1640 1645 1650 Val Ser As n Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr I l e Ser 1655 1660 1665 Lys Al a Lys Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o 1670 1675 1680 Pr o Ser Ar g As p Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys 1685 1690 1695 Leu Val Lys Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u 1700 1705 1710 Ser As n Gl y Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val 1715 1720 1725

   Page 466

   97047_1

   2018203206 08 May 2018

   Leu As p 1730 Ser As p Gl y Ser Phe 1735 Phe Leu Tyr Ser Lys 1740 Leu Thr Val As p Lys Ser Arg Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val 1745 1750 1755 Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser 1760 1765 1770 Leu Ser Pr o Gl y Lys

   1775 <210> 120 <211> 1565 <212> PRT <213> Artificial Sequence <220>

   <223> pSYN VWF 036 pr ot ei n sequence <400> 120

   Met I l e Pr o Al a Ar g Phe Al a Gl y Val Leu Leu Al a Leu Al a Leu I l e 1 5 10 15

   Leu Pr o Gl y Thr Leu Cy s Al a Gl u Gl y Thr Ar g Gl y Ar g Ser Ser Thr 20 25 30

   Page 467

   97047_1

   2018203206 08 May 2018

   Al a Ar g Cy s Ser Leu Phe Gl y Ser 40 Asp Phe Val As n Thr 45 Phe As p Gl y 35 Ser Met Ty r Ser Phe Al a Gl y Ty r Cy s Ser Ty r Leu Leu Al a Gl y Gl y 50 55 60 Cy s Gl n Lys Ar g Ser Phe Ser Ile Ile Gl y As p Phe Gl n As n Gl y Lys 65 70 75 80 Ar g Val Ser Leu Ser Val Ty r Leu Gl y Gl u Phe Phe As p I l e Hi s Leu 85 90 95 Phe Val As n Gl y Thr Val Thr Gl n Gl y As p Gl n Ar g Val Ser Met Pr o 100 105 110 Ty r Al a Ser Lys Gl y Leu Ty r Leu Gl u Thr Gl u Al a Gl y Ty r Ty r Lys 115 120 125 Leu Ser Gl y Gl u Al a Ty r Gl y Phe Val Al a Ar g Ile As p Gl y Ser Gl y 130 135 140 As n Phe Gl n Val Leu Leu Ser As p Ar g Ty r Phe As n Lys Thr Cy s Gl y 145 150 155 160 Leu Cy s Gl y As n Phe As n Ile Phe Al a Gl u As p As p Phe Met Thr Gl n

   Page 468

   2018203206 08 May 2018

   97047_1

   165 170 175

   Gl u Gl y Thr Leu 180 Thr Ser As p Pr o Tyr 185 As p Phe Al a As n Ser 190 Tr p Al a Leu Ser Ser Gl y Gl u Gl n Tr p Cys Gl u Ar g Al a Ser Pr o Pr o Ser Ser 195 200 205 Ser Cys As n I l e Ser Ser Gl y Gl u Met Gl n Lys Gl y Leu Tr p Gl u Gl n 210 215 220 Cys Gl n Leu Leu Lys Ser Thr Ser Val Phe Al a Ar g Cys Hi s Pr o Leu 225 230 235 240 Val As p Pr o Gl u Pr o Phe Val Al a Leu Cys Gl u Lys Thr Leu Cys Gl u 245 250 255 Cys Al a Gl y Gl y Leu Gl u Cys Al a Cys Pr o Al a Leu Leu Gl u Tyr Al a 260 265 270 Ar g Thr Cys Al a Gl n Gl u Gl y Met Val Leu Tyr Gl y Tr p Thr As p Hi s 275 280 285 Ser Al a Cys Ser Pr o Val Cys Pr o Al a Gl y Met Gl u Tyr Ar g Gl n Cys 290 295 300

   Page 469

   97047_1

   2018203206 08 May 2018

   Val 305 Ser Pr o Cy s Al a Ar g 310 Thr Cy s Gl n Ser Leu 315 Hi s I l e As n Gl u Met 320 Cy s Gl n Gl u Ar g Cy s Val As p Gl y Cy s Ser Cy s Pr o Gl u Gl y Gl n Leu 325 330 335 Leu As p Gl u Gl y Leu Cy s Val Gl u Ser Thr Gl u Cy s Pr o Cy s Val Hi s 340 345 350 Ser Gl y Lys Ar g Ty r Pr o Pr o Gl y Thr Ser Leu Ser Ar g As p Cy s As n 355 360 365 Thr Cy s I l e Cy s Ar g As n Ser Gl n Tr p Ile Cy s Ser As n Gl u Gl u Cy s 370 375 380 Pr o Gl y Gl u Cy s Leu Val Thr Gl y Gl n Ser Hi s Phe Lys Ser Phe As p 385 390 395 400 As n Ar g Ty r Phe Thr Phe Ser Gl y Ile Cy s Gl n Ty r Leu Leu Al a Ar g 405 410 415 As p Cy s Gl n As p Hi s Ser Phe Ser Ile Val Ile Gl u Thr Val Gl n Cy s 420 425 430 Al a As p As p Ar g As p Al a Val Cy s Thr Ar g Ser Val Thr Val Ar g Leu

   Page 470

   2018203206 08 May 2018

   97047_1

   435 440 445

   Pr o Gl y 450 Leu Hi s As n Ser Leu 455 Val Lys Leu Lys Hi s 460 Gl y Al a Gl y Val Al a Met As p Gl y Gl n As p Ile Gl n Leu Pr o Leu Leu Lys Gl y As p Leu 465 470 475 480 Ar g I l e Gl n Hi s Thr Val Thr Al a Ser Val Ar g Leu Ser Tyr Gl y Gl u 485 490 495 As p Leu Gl n Met As p Tr p As p Gl y Ar g Gl y Ar g Leu Leu Val Lys Leu 500 505 510 Ser Pr o Val Tyr Al a Gl y Lys Thr Cys Gl y Leu Cys Gl y As n Tyr As n 515 520 525 Gl y As n Gl n Gl y As p As p Phe Leu Thr Pr o Ser Gl y Leu Al a Gl u Pr o 530 535 540 Ar g Val Gl u As p Phe Gl y As n Al a Tr p Lys Leu Hi s Gl y As p Cys Gl n 545 550 555 560 As p Leu Gl n Lys Gl n Hi s Ser As p Pr o Cys Al a Leu As n Pr o Ar g Met 565 570 575

   Page 471

   97047_1

   2018203206 08 May 2018

   Thr Ar g Phe Ser 580 Gl u Gl u Al a Cys Al a 585 Val Leu Thr Ser Pr o 590 Thr Phe Gl u Al a Cys 595 Hi s Ar g Al a Val Ser 600 Pr o Leu Pr o Tyr Leu 605 Ar g As n Cys Ar g Tyr 610 As p Val Cys Ser Cys 615 Ser As p Gl y Ar g Gl u 620 Cys Leu Cys Gl y Al a 625 Leu Al a Ser Tyr Al a 630 Al a Al a Cys Al a Gl y 635 Ar g Gl y Val Ar g Val 640 Al a Tr p Ar g Gl u Pr o 645 Gl y Ar g Cys Gl u Leu 650 As n Cys Pr o Lys Gl y 655 Gl n Val Tyr Leu Gl n 660 Cys Gl y Thr Pr o Cys 665 As n Leu Thr Cys Ar g 670 Ser Leu Ser Tyr Pr o 675 As p Gl u Gl u Cys As n 680 Gl u Al a Cys Leu Gl u 685 Gl y Cys Phe Cys Pr o 690 Pr o Gl y Leu Tyr Met 695 As p Gl u Ar g Gl y As p 700 Cys Val Pr o Lys Al a Gl n Cys Pr o Cys Tyr Tyr As p Gl y Gl u Ile Phe Gl n Pr o Gl u As p

   Page 472

   2018203206 08 May 2018

   97047_1

   705 710 715 720

   I l e Phe Ser Asp Hi s 725 Hi s Thr Met Cys Tyr 730 Cys Gl u As p Gl y Phe 735 Met Hi s Cys Thr Met Ser Gl y Val Pr o Gl y Ser Leu Leu Pr o As p Al a Val 740 745 750 Leu Ser Ser Pr o Leu Ser Hi s Ar g Ser Lys Ar g Ser Leu Ser Cys Ar g 755 760 765 Pr o Pr o Met Val Lys Leu Val Cys Pr o Al a As p As n Leu Ar g Al a Gl u 770 775 780 Gly Leu Gl u Cys Thr Lys Thr Cys Gl n As n Tyr As p Leu Gl u Cys Met 785 790 795 800 Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o Pr o Gl y Met Val Ar g 805 810 815 Hi s Gl u Asn Ar g Cys Val Al a Leu Gl u Ar g Cys Pr o Cys Phe Hi s Gl n 820 825 830 Gl y Lys Gl u Tyr Al a Pr o Gl y Gl u Thr Val Lys Ile Gl y Cys As n Thr 835 840 845

   Page 473

   97047_1

   2018203206 08 May 2018

   Cys Val Cys Ar g As p Ar g Lys 855 Trp Asn Cys Thr As p 860 Hi s Val Cys As p 850 Al a Thr Cys Ser Thr Ile Gl y Met Al a Hi s Tyr Leu Thr Phe As p Gl y 865 870 875 880 Leu Lys Tyr Leu Phe Pr o Gl y Gl u Cys Gl n Tyr Val Leu Val Gl n As p 885 890 895 Tyr Cys Gl y Ser As n Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys 900 905 910 Gl y Cys Ser Hi s Pr o Ser Val Lys Cys Lys Lys Ar g Val Thr I l e Leu 915 920 925 Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y Gl u Val As n Val Lys 930 935 940 Ar g Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g 945 950 955 960 Tyr I l e I l e Leu Leu Leu Gl y Lys Al a Leu Ser Val Val Tr p As p Ar g 965 970 975 Hi s Leu Ser I l e Ser Val Val Leu Lys Gl n Thr Tyr Gl n Gl u Lys Val

   Page 474

   980

   2018203206 08 May 2018

   97047_1

   985 990

   Cys Gl y Leu Cys Gl y Asn Phe Asp Gl y I l e Gl n Asn Asn Asp Leu Thr 995 1000 1005

   Ser Ser 1010 As n Leu Gl n Val Gl u 1015 Gl u As p Pr o Val As p 1020 Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cys Al a As p Thr Ar g Lys Val Pr o 1025 1030 1035 Leu As p Ser Ser Pr o Al a Thr Cys Hi s As n As n Ile Met Lys Gl n 1040 1045 1050 Thr Met Val As p Ser Ser Cys Ar g Ile Leu Thr Ser As p Val Phe 1055 1060 1065 Gl n As p Cys As n Lys Leu Val As p Pr o Gl u Pr o Tyr Leu As p Val 1070 1075 1080 Cys I l e Tyr As p Thr Cys Ser Cys Gl u Ser Ile Gl y As p Cys Al a 1085 1090 1095 Al a Phe Cys As p Thr Ile Al a Al a Tyr Al a Hi s Val Cys Al a Gl n 1100 1105 1110

   Page 475

   97047_1

   2018203206 08 May 2018

   Hi s Gl y 1115 Lys Val Val Thr Tr p 1120 Ar g Thr Al a Thr Leu 1125 Cy s Pr o Gl n Ser Cy s Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Ty r Gl u Al a Gl u 1130 1135 1140 Tr p Ar g Ty r As n Ser Cy s Al a Pr o Al a Cy s Gl n Val Thr Cy s Gl n 1145 1150 1155 Hi s Pr o Gl u Pr o Leu Al a Cy s Pr o Val Gl n Cy s Val Gl u Gl y Cy s 1160 1165 1170 Hi s Al a Hi s Cy s Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu Leu Gl n 1175 1180 1185 Thr Cy s Val As p Pr o Gl u As p Cy s Pr o Val Cy s Gl u Val Al a Gl y 1190 1195 1200 Ar g Ar g Phe Al a Ser Gl y Lys Lys Val Thr Leu As n Pr o Ser As p 1205 1210 1215 Pr o Gl u Hi s Cy s Gl n Ile Cy s Hi s Cy s As p Val Val As n Leu Thr 1220 1225 1230 Cy s Gl u Al a Cy s Gl n Gl u Pr o Ile Ser Gl y Gl y Gl y Gl y Ser Gl y

   Page 476

   97047_1

   2018203206 08 May 2018

   1235

   1240

   1245

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1250 1255 1260

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1265 1270 1275

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1280 1285 1290

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1295 1300 1305

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Leu 1310 1315 1320

   Val Pr o Ar g Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser 1325 1330 1335

   Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1340 1345 1350

   Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys Asp Thr

   1355 1360 1365

   Page 477

   97047_1

   2018203206 08 May 2018

   Leu Met 1370 I l e Ser Ar g Thr Pr o 1375 Gl u Val Thr Cys Val 1380 Val Val As p Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Tyr Val As p 1385 1390 1395 Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n 1400 1405 1410 Tyr As n Ser Thr Tyr Ar g Val Val Ser Val Leu Thr Val Leu Hi s 1415 1420 1425 Gl n As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n 1430 1435 1440 Lys Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a Lys 1445 1450 1455 Gl y Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g 1460 1465 1470 As p Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys 1475 1480 1485 Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y

   Page 478

   2018203206 08 May 2018

   1490 1495 97047 _1 1500 Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser 1505 1510 1515 As p Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser 1520 1525 1530 Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u 1535 1540 1545 Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser Leu Ser Leu Ser Pr o 1550 1555 1560

   Gl y Lys 1565 <210> 121 <211> 247 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYN Fc-015 protein sequence <400> 121

   Met Gl u Thr Asp Thr Leu Leu Leu Tr p Val Leu Leu Leu Tr p Val Pr o

   Page 479

   97047_1

   2018203206 08 May 2018

   1 5 10 15 Gl y Ser Thr Gl y As p Lys Thr Hi s Thr Cy s Pr o Pr o Cy s Pr o Al a Pr o 20 25 30 Gl u Leu Leu Gl y Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys 35 40 45 Asp Thr Leu Met Ile Ser Ar g Thr Pr o Gl u Val Thr Cy s Val Val Val 50 55 60 Asp Val Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Ty r Val As p 65 70 75 80 Gl y Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Ty r 85 90 95 As n Ser Thr Ty r Ar g Val Val Ser Val Leu Thr Val Leu Hi s Gl n As p 100 105 110 Tr p Leu As n Gl y Lys Gl u Ty r Lys Cys Lys Val Ser As n Lys Al a Leu 115 120 125 Pr o Al a Pr o I l e Gl u Lys Thr Ile Ser Lys Al a Lys Gl y Gl n Pr o Ar g 130 135 140

   Page 480

   97047_1

   2018203206 08 May 2018

   Gl u 145 Pr o Gl n Val Tyr Thr 150 Leu Pr o Pr o Ser Ar g 155 As p Gl u Leu Thr Lys 160 As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y Phe Tyr Pr o Ser As p 165 170 175 I l e Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Tyr Lys 180 185 190 Thr Thr Pr o Pr o Val Leu As p Ser As p Gl y Ser Phe Phe Leu Tyr Ser 195 200 205 Lys Leu Thr Val As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe Ser 210 215 220 Cys Ser Val Met Hi s Gl u Al a Leu Hi s As n Hi s Tyr Thr Gl n Lys Ser 225 230 235 240

   Leu Ser Leu Ser

   Pr o Gl y Lys

   245

   <210> 122 <211> 33 <212> PRT <213> Art i f i ci al Sequence

   Page 481

   2018203206 08 May 2018

   97047_1 <220>

   <223> pol ypept i de

   <400> 122 Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1 5 10 15

   Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Leu Val Pr o Ar g Gl y Ser Gl y 20 25 30

   Gl y <210> 123 <211> 1240 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> VWF-D1D2DD3 <400> 123

   Met I l e Pr o Al a Ar g Phe Al a Gl y Val Leu Leu Al a Leu Al a Leu I l e

   1 5 10 15

   Leu Pr o Gl y Thr Leu Cy s Al a Gl u Gl y Thr Ar g Gl y Ar g Ser Ser Thr

   20 25 30

   Page 482

   97047_1

   2018203206 08 May 2018

   Al a Arg Cys Ser Leu Phe Gl y Ser 40 Asp Phe Val As n Thr 45 Phe As p Gl y 35 Ser Met Tyr Ser Phe Al a Gl y Tyr Cys Ser Tyr Leu Leu Al a Gl y Gl y 50 55 60 Cys Gl n Lys Ar g Ser Phe Ser Ile Ile Gl y As p Phe Gl n As n Gl y Lys 65 70 75 80 Ar g Val Ser Leu Ser Val Tyr Leu Gl y Gl u Phe Phe As p I l e Hi s Leu 85 90 95 Phe Val As n Gl y Thr Val Thr Gl n Gl y As p Gl n Ar g Val Ser Met Pr o 100 105 110 Tyr Al a Ser Lys Gl y Leu Tyr Leu Gl u Thr Gl u Al a Gl y Tyr Tyr Lys 115 120 125 Leu Ser Gl y Gl u Al a Tyr Gl y Phe Val Al a Ar g Ile As p Gl y Ser Gl y 130 135 140 As n Phe Gl n Val Leu Leu Ser As p Ar g Tyr Phe As n Lys Thr Cys Gl y 145 150 155 160 Leu Cys Gl y As n Phe As n Ile Phe Al a Gl u As p As p Phe Met Thr Gl n

   Page 483

   2018203206 08 May 2018

   97047_1

   165 170 175

   Gl u Gl y Thr Leu 180 Thr Ser As p Pr o Tyr 185 As p Phe Al a As n Ser 190 Tr p Al a Leu Ser Ser Gl y Gl u Gl n Tr p Cys Gl u Ar g Al a Ser Pr o Pr o Ser Ser 195 200 205 Ser Cys As n I l e Ser Ser Gl y Gl u Met Gl n Lys Gl y Leu Tr p Gl u Gl n 210 215 220 Cys Gl n Leu Leu Lys Ser Thr Ser Val Phe Al a Ar g Cys Hi s Pr o Leu 225 230 235 240 Val As p Pr o Gl u Pr o Phe Val Al a Leu Cys Gl u Lys Thr Leu Cys Gl u 245 250 255 Cys Al a Gl y Gl y Leu Gl u Cys Al a Cys Pr o Al a Leu Leu Gl u Tyr Al a 260 265 270 Ar g Thr Cys Al a Gl n Gl u Gl y Met Val Leu Tyr Gl y Tr p Thr As p Hi s 275 280 285 Ser Al a Cys Ser Pr o Val Cys Pr o Al a Gl y Met Gl u Tyr Ar g Gl n Cys 290 295 300

   Page 484

   97047 1

   2018203206 08 May 2018

   Val 305 Ser Pr o Cy s Al a Ar g 310 Thr Cy s Gl n Ser Leu 315 Hi s I l e As n Gl u Met 320 Cy s Gl n Gl u Ar g Cy s Val As p Gl y Cy s Ser Cy s Pr o Gl u Gl y Gl n Leu 325 330 335 Leu As p Gl u Gl y Leu Cy s Val Gl u Ser Thr Gl u Cy s Pr o Cy s Val Hi s 340 345 350 Ser Gl y Lys Ar g Ty r Pr o Pr o Gl y Thr Ser Leu Ser Ar g As p Cy s As n 355 360 365 Thr Cy s I l e Cy s Ar g As n Ser Gl n Tr p Ile Cy s Ser As n Gl u Gl u Cy s 370 375 380 Pr o Gl y Gl u Cy s Leu Val Thr Gl y Gl n Ser Hi s Phe Lys Ser Phe As p 385 390 395 400 As n Ar g Ty r Phe Thr Phe Ser Gl y Ile Cy s Gl n Ty r Leu Leu Al a Ar g 405 410 415 As p Cy s Gl n As p Hi s Ser Phe Ser Ile Val Ile Gl u Thr Val Gl n Cy s 420 425 430 Al a As p As p Ar g As p Al a Val Cy s Thr Ar g Ser Val Thr Val Ar g Leu

   Page 485

   2018203206 08 May 2018

   97047_1

   435 440 445

   Pr o Gl y 450 Leu Hi s As n Ser Leu 455 Val Lys Leu Lys Hi s 460 Gl y Al a Gl y Val Al a Met As p Gl y Gl n As p Ile Gl n Leu Pr o Leu Leu Lys Gl y As p Leu 465 470 475 480 Ar g I l e Gl n Hi s Thr Val Thr Al a Ser Val Ar g Leu Ser Tyr Gl y Gl u 485 490 495 As p Leu Gl n Met As p Tr p As p Gl y Ar g Gl y Ar g Leu Leu Val Lys Leu 500 505 510 Ser Pr o Val Tyr Al a Gl y Lys Thr Cys Gl y Leu Cys Gl y As n Tyr As n 515 520 525 Gl y As n Gl n Gl y As p As p Phe Leu Thr Pr o Ser Gl y Leu Al a Gl u Pr o 530 535 540 Ar g Val Gl u As p Phe Gl y As n Al a Tr p Lys Leu Hi s Gl y As p Cys Gl n 545 550 555 560 As p Leu Gl n Lys Gl n Hi s Ser As p Pr o Cys Al a Leu As n Pr o Ar g Met 565 570 575

   Page 486

   97047_1

   2018203206 08 May 2018

   Thr Ar g Phe Ser 580 Gl u Gl u Al a Cy s Al a 585 Val Leu Thr Ser Pr o 590 Thr Phe Gl u Al a Cy s 595 Hi s Ar g Al a Val Ser 600 Pr o Leu Pr o Ty r Leu 605 Ar g As n Cy s Ar g Ty r 610 As p Val Cy s Ser Cy s 615 Ser As p Gl y Ar g Gl u 620 Cy s Leu Cy s Gl y Al a 625 Leu Al a Ser Ty r Al a 630 Al a Al a Cy s Al a Gl y 635 Ar g Gl y Val Ar g Val 640 Al a Tr p Ar g Gl u Pr o 645 Gl y Ar g Cy s Gl u Leu 650 As n Cy s Pr o Lys Gl y 655 Gl n Val Ty r Leu Gl n 660 Cy s Gl y Thr Pr o Cy s 665 As n Leu Thr Cy s Ar g 670 Ser Leu Ser Ty r Pr o 675 As p Gl u Gl u Cy s As n 680 Gl u Al a Cy s Leu Gl u 685 Gl y Cy s Phe Cy s Pr o 690 Pr o Gl y Leu Ty r Met 695 As p Gl u Ar g Gl y As p 700 Cy s Val Pr o Lys Al a Gl n Cy s Pr o Cy s Ty r Ty r As p Gl y Gl u Ile Phe Gl n Pr o Gl u As p

   Page 487

   2018203206 08 May 2018

   97047_1

   705 710 715 720

   I l e Phe Ser As p Hi s 725 Hi s Thr Met Cys Tyr 730 Cys Gl u As p Gl y Phe 735 Met Hi s Cys Thr Met Ser Gl y Val Pr o Gl y Ser Leu Leu Pr o As p Al a Val 740 745 750 Leu Ser Ser Pr o Leu Ser Hi s Ar g Ser Lys Ar g Ser Leu Ser Cys Ar g 755 760 765 Pr o Pr o Met Val Lys Leu Val Cys Pr o Al a As p As n Leu Ar g Al a Gl u 770 775 780 Gl y Leu Gl u Cys Thr Lys Thr Cys Gl n As n Tyr As p Leu Gl u Cys Met 785 790 795 800 Ser Met Gl y Cys Val Ser Gl y Cys Leu Cys Pr o Pr o Gl y Met Val Ar g 805 810 815 Hi s Gl u As n Ar g Cys Val Al a Leu Gl u Ar g Cys Pr o Cys Phe Hi s Gl n 820 825 830 Gl y Lys Gl u Tyr Al a Pr o Gl y Gl u Thr Val Lys Ile Gl y Cys As n Thr 835 840 845

   Page 488

   97047_1

   2018203206 08 May 2018

   Cys Val Cy s Ar g As p Ar g Lys 855 Tr p As n Cy s Thr As p 860 Hi s Val Cy s As p 850 Al a Thr Cy s Ser Thr Ile Gl y Met Al a Hi s Ty r Leu Thr Phe As p Gl y 865 870 875 880 Leu Lys Ty r Leu Phe Pr o Gl y Gl u Cy s Gl n Ty r Val Leu Val Gl n As p 885 890 895 Ty r Cy s Gl y Ser As n Pr o Gl y Thr Phe Ar g Ile Leu Val Gl y As n Lys 900 905 910 Gl y Cy s Ser Hi s Pr o Ser Val Lys Cy s Lys Lys Ar g Val Thr I l e Leu 915 920 925 Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y Gl u Val As n Val Lys 930 935 940 Ar g Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val Val Gl u Ser Gl y Ar g 945 950 955 960 Ty r I l e I l e Leu Leu Leu Gl y Lys Al a Leu Ser Val Val Tr p As p Ar g 965 970 975 Hi s Leu Ser I l e Ser Val Val Leu Lys Gl n Thr Ty r Gl n Gl u Lys Val

   Page 489

   980

   2018203206 08 May 2018

   97047_1

   985 990

   Cys Gl y Leu Cys Gl y Asn Phe Asp Gl y Ile Gl n Asn Asn Asp Leu Thr 995 1000 1005

   Ser Ser 1010 As n Leu Gl n Val Gl u 1015 Gl u As p Pr o Val As p 1020 Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cys Al a As p Thr Ar g Lys Val Pr o 1025 1030 1035 Leu As p Ser Ser Pr o Al a Thr Cys Hi s As n As n Ile Met Lys Gl n 1040 1045 1050 Thr Met Val As p Ser Ser Cys Ar g Ile Leu Thr Ser As p Val Phe 1055 1060 1065 Gl n As p Cys As n Lys Leu Val As p Pr o Gl u Pr o Tyr Leu As p Val 1070 1075 1080 Cys I l e Tyr As p Thr Cys Ser Cys Gl u Ser Ile Gl y As p Cys Al a 1085 1090 1095 Cys Phe Cys As p Thr Ile Al a Al a Tyr Al a Hi s Val Cys Al a Gl n 1100 1105 1110

   Page 490

   97047_1

   2018203206 08 May 2018

   Hi s Gl y 1115 Lys Val Val Thr Tr p 1120 Ar g Thr Al a Thr Leu 1125 Cys Pr o Gl n Ser Cys Gl u Gl u Ar g As n Leu Ar g Gl u As n Gl y Tyr Gl u Cys Gl u 1130 1135 1140 Tr p Ar g Tyr As n Ser Cys Al a Pr o Al a Cys Gl n Val Thr Cys Gl n 1145 1150 1155 Hi s Pr o Gl u Pr o Leu Al a Cys Pr o Val Gl n Cys Val Gl u Gl y Cys 1160 1165 1170 Hi s Al a Hi s Cys Pr o Pr o Gl y Lys Ile Leu As p Gl u Leu Leu Gl n 1175 1180 1185 Thr Cys Val As p Pr o Gl u As p Cys Pr o Val Cys Gl u Val Al a Gl y 1190 1195 1200 Ar g Ar g Phe Al a Ser Gl y Lys Lys Val Thr Leu As n Pr o Ser As p 1205 1210 1215 Pr o Gl u Hi s Cys Gl n Ile Cys Hi s Cys As p Val Val As n Leu Thr 1220 1225 1230 Cys Gl u Al a Cys Gl n Gl u Pr o

   Page 491

   97047_1

   2018203206 08 May 2018

   1235 1240 <210> 124 <211> 477 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> VWF- D D3 <400> 124

   Ser 1 Leu Ser Cys Ar g 5 Pr o Pr o Met As n Leu Ar g Al a 20 Gl u Gl y Leu Gl u As p Leu Gl u 35 Cys Met Ser Met Gl y 40 Pr o Gl y 50 Met Val Ar g Hi s Gl u 55 As n Pr o 65 Cys Phe Hi s Gl n Gl y 70 Lys Gl u I l e Gl y Cys As n Thr Cys Val Cys

   Val Lys 10 Leu Val Cys Pr o Al a 15 As p Cys 25 Thr Lys Thr Cys Gl n 30 As n Tyr Cys Val Ser Gl y Cys 45 Leu Cys Pr o Ar g Cys Val Al a 60 Leu Gl u Ar g Cys Tyr Al a Pr o 75 Gl y Gl u Thr Val Lys 80 Ar g As p Ar g Lys Tr p As n Cys Thr

   Page 492

   2018203206 08 May 2018

   97047_1

   85 90 95

   As p Hi s Val Cys 100 As p Al a Thr Cys Ser 105 Thr Ile Gl y Met Al a 110 Hi s Tyr Leu Thr Phe As p Gl y Leu Lys Tyr Leu Phe Pr o Gl y Gl u Cys Gl n Tyr 115 120 125 Val Leu Val Gl n As p Tyr Cys Gl y Ser As n Pr o Gl y Thr Phe Ar g I l e 130 135 140 Leu Val Gl y As n Lys Gl y Cys Ser Hi s Pr o Ser Val Lys Cys Lys Lys 145 150 155 160 Ar g Val Thr I l e Leu Val Gl u Gl y Gl y Gl u Ile Gl u Leu Phe As p Gl y 165 170 175 Gl u Val As n Val Lys Ar g Pr o Met Lys As p Gl u Thr Hi s Phe Gl u Val 180 185 190 Val Gl u Ser Gl y Ar g Tyr Ile Ile Leu Leu Leu Gl y Lys Al a Leu Ser 195 200 205 Val Val Tr p As p Ar g Hi s Leu Ser Ile Ser Val Val Leu Lys Gl n Thr 210 215 220

   Page 493

   97047_1

   2018203206 08 May 2018

   Ty r 225 Gl n Gl u Lys Val Cy s 230 Gl y Leu Cy s Gl y As n 235 Phe As p Gl y I l e Gl n 240 As n As n As p Leu Thr Ser Ser As n Leu Gl n Val Gl u Gl u As p Pr o Val 245 250 255 As p Phe Gl y As n Ser Tr p Lys Val Ser Ser Gl n Cy s Al a As p Thr Ar g 260 265 270 Lys Val Pr o Leu As p Ser Ser Pr o Al a Thr Cy s Hi s As n As n I l e Met 275 280 285 Lys Gl n Thr Met Val As p Ser Ser Cy s Ar g Ile Leu Thr Ser As p Val 290 295 300 Phe Gl n As p Cy s As n Lys Leu Val As p Pr o Gl u Pr o Ty r Leu As p Val 305 310 315 320 Cy s I l e Ty r As p Thr Cy s Ser Cy s Gl u Ser Ile Gl y As p Cy s Al a Cy s 325 330 335 Phe Cy s As p Thr Ile Al a Al a Ty r Al a Hi s Val Cy s Al a Gl n Hi s Gl y 340 345 350 Lys Val Val Thr Tr p Ar g Thr Al a Thr Leu Cy s Pr o Gl n Ser Cy s Gl u

   Page 494

   2018203206 08 May 2018

   355 360 97047_1 365 Gl u Ar g As n Leu Ar g Gl u As n Gl y Tyr Gl u Cys Gl u Tr p Ar g Tyr As n 370 375 380 Ser Cys Al a Pr o Al a Cys Gl n Val Thr Cys Gl n Hi s Pr o Gl u Pr o Leu 385 390 395 400 Al a Cys Pr o Val Gl n Cys Val Gl u Gl y Cys Hi s Al a Hi s Cys Pr o Pr o 405 410 415 Gl y Lys I l e Leu As p Gl u Leu Leu Gl n Thr Cys Val As p Pr o Gl u As p 420 425 430 Cys Pr o Val Cys Gl u Val Al a Gl y Ar g Ar g Phe Al a Ser Gl y Lys Lys 435 440 445 Val Thr Leu As n Pr o Ser As p Pr o Gl u Hi s Cys Gl n I l e Cys Hi s Cys 450 455 460

   Asp Val Val Asn Leu Thr Cys Gl u Al a Cys Gl n Gl u Pro

   465

   470

   475 <210> 125 <211> 5055 <212> DNA

   Page 495

   2018203206 08 May 2018

   97047_1 <213> Artificial Sequence <220>

   <223> pSYNFVI I I 010

   <400> 125 at gcaaat ag agct ct ccac ct gct t ct t t ct gt gcct t t t gcgat t ct g ct t t agt gcc 60 accagaagat act acct ggg t gcagt ggaa ct gt cat ggg act at at gca aagt gat ct c 120 ggt gagct gc ct gt ggacgc aagat t t cct cct agagt gc caaaat ct t t t ccat t caac 180 acct cagt cg t gt acaaaaa gact ct gt 11 gt agaat t ca cggat cacct 111 caacat c 240 gct aagccaa ggccaccct g gat gggt ct g ct aggt cct a ccat ccaggc tgaggtttat 300 gat acagt gg t cat t acact t aagaacat g gct t cccat c ct gt cagt ct t cat gct gt t 360 ggt gt at cct act ggaaagc tt ctgaggga gct gaat at g at gat cagac cagt caaagg 420 gagaaagaag at gat aaagt ct t ccct ggt ggaagccat a cat at gt ct g gcaggt cct g 480 aaagagaat g gt ccaat ggc ct ct gaccca ct gt gcct t a cct act cat a t ct t t ct cat 540 gt ggacct gg t aaaagact t gaat t caggc ct cat t ggag ccct act agt at gt agagaa 600 gggagt ct gg ccaaggaaaa gacacagacc 11 gcacaaat 11 at act act t t t t gct gt a 660 t t t gat gaag ggaaaagt t g gcact cagaa acaaagaact cct t gat gca ggat agggat 720 gct gcat ct g ct cgggcct g gcct aaaat g cacacagt ca at ggt t at gt aaacaggt ct 780 ct gccaggt c t gat t ggat g ccacaggaaa t cagt ct at t ggcat gt gat t ggaat gggc 840

   Page 496

   2018203206 08 May 2018

   accact cct g aagt gcact c aat at t cct c 97047_1 gaaggt caca cat t t ct t gt gaggaaccat 900 cgccaggcgt cct t ggaaat ct cgccaat a act t t cct t a ct gct caaac act ct t gat g 960 gacct t ggac agt t t ct act gt t t t gt cat at ct ct t ccc accaacat ga t ggcat ggaa 1020 gct t at gt ca aagt agacag ct gt ccagag gaaccccaac t acgaat gaa aaat aat gaa 1080 gaagcggaag act at gat ga t gat ct t act gat t ct gaaa t ggat gt ggt caggt t t gat 1140 gat gacaact ct cct t cct t t at ccaaat t cgct cagt t g ccaagaagca t cct aaaact 1200 t gggt acat t acat t gct gc t gaagaggag gact gggact at gct ccct t agt cct cgcc 1260 cccgat gaca gaagt t at aa aagt caat at t t gaacaat g gccct cagcg gat t ggt agg 1320 aagt acaaaa aagt ccgat t t at ggcat ac acagat gaaa cct t t aagac t cgt gaagct 1380 at t cagcat g aat caggaat ct t gggacct t t act t t at g gggaagt t gg agacacact g 1440 t t gat t at at t t aagaat ca agcaagcaga ccat at aaca t ct accct ca cggaat cact 1500 gat gt ccgt c ct t t gt at t c aaggagat t a ccaaaaggt g t aaaacat t t gaaggat t t t 1560 ccaat t ct gc caggagaaat at t caaat at aaat ggacag t gact gt aga agat gggcca 1620 act aaat cag at cct cggt g cct gacccgc t at t act ct a gt t t cgt t aa t at ggagaga 1680 gat ct agct t caggact cat t ggccct ct c ct cat ct gct acaaagaat c t gt agat caa 1740 agaggaaacc agat aat gt c agacaagagg aat gt cat cc t gt t t t ct gt at t t gat gag 1800 aaccgaagct ggt acct cac agagaat at a caacgct 11 c t ccccaat cc agct ggagt g 1860

   Page 497

   2018203206 08 May 2018

   cagct t gagg at ccagagt t ccaagcct cc 97047_1 aacat cat gc acagcat caa tggct at gtt 1920 t t t gat agt t t gcagt t gt c agt t t gt t t g cat gaggt gg cat act ggt a cat t ct aagc 1980 at t ggagcac agact gact t cct t t ct gt c 11 ct t ct ct g gat at acct t caaacacaaa 2040 at ggt ct at g aagacacact caccct at t c ccat t ct cag gagaaact gt ct t cat gt cg 2100 at ggaaaacc caggt ct at g gat t ct gggg t gccacaact cagact t t cg gaacagaggc 2160 at gaccgcct t act gaaggt 11 ct agt t gt gacaagaaca ct ggt gat t a t t acgaggac 2220 agt t at gaag at at t t cagc at act t gct g agt aaaaaca at gccat t ga accaagaagc 2280 t t ct ct caaa acccaccagt ct t gaaacgc cat caacggg aaat aact cg t act act ct t 2340 cagt cagat c aagaggaaat t gact at gat gat accat at cagt t gaaat gaagaaggaa 2400 gat t t t gaca t t t at gat ga ggat gaaaat cagagccccc gcagct t t ca aaagaaaaca 2460 cgacact at t t t at t gct gc agt ggagagg ct ct gggat t at gggat gag t agct cccca 2520 cat gt t ct aa gaaacagggc t cagagt ggc agt gt ccct c agt t caagaa agt t gt t t t c 2580 caggaat t t a ct gat ggct c ct 11 act cag ccct t at acc gt ggagaact aaat gaacat 2640 t t gggact cc t ggggccat a t at aagagca gaagt t gaag at aat at cat ggt aact t t c 2700 agaaat cagg cct ct cgt cc ct at t cct t c t at t ct agcc 11 at 11 ct t a t gaggaagat 2760 cagaggcaag gagcagaacc t agaaaaaac 111 gt caagc ct aat gaaac caaaact t ac 2820 t t t t ggaaag t gcaacat ca t at ggcaccc act aaagat g agt t t gact g caaagcct gg 2880

   Page 498

   2018203206 08 May 2018

   gct t at 11 ct ct gat gt t ga cct ggaaaaa 97047_1 gat gt gcact caggcct gat t ggacccct t 2940 ct ggt ct gcc acact aacac act gaaccct gct cat ggga gacaagt gac agt acaggaa 3000 t t t gct ct gt t t t t caccat ct t t gat gag accaaaagct ggt act t cac t gaaaat at g 3060 gaaagaaact gcagggct cc ct gcaat at c cagat ggaag at cccact t t t aaagagaat 3120 t at cgct t cc at gcaat caa t ggct acat a at ggat acac t acct ggct t agt aat ggct 3180 caggat caaa ggat t cgat g gt at ct gct c agcat gggca gcaat gaaaa cat ccat t ct 3240 at t cat t t ca gt ggacat gt gt t cact gt a cgaaaaaaag aggagt at aa aat ggcact g 3300 t acaat ct ct at ccaggt gt 1111 gagaca gt ggaaat gt t accat ccaa agct ggaat t 3360 t ggcgggt gg aat gcct t at t ggcgagcat ct acat gct g ggat gagcac act t t t t ct g 3420 gt gt acagca at aagt gt ca gact cccct g ggaat ggct t ct ggacacat t agagat t t t 3480 cagat t acag ct t caggaca at at ggacag t gggccccaa agct ggccag act t cat t at 3540 t ccggat caa t caat gcct g gagcaccaag gagccct t t t ct t ggat caa ggt ggat ct g 3600 t t ggcaccaa t gat t at t ca cggcat caag acccagggt g cccgt cagaa gt t ct ccagc 3660 ct ct acat ct ct cagt t t at cat cat gt at agt ct t gat g ggaagaagt g gcagact t at 3720 cgaggaaat t ccact ggaac ct t aat ggt c t t ct t t ggca at gt ggat t c at ct gggat a 3780 aaacacaat a t t t t t aaccc t ccaat t at t gct cgat aca t ccgt t t gca cccaact cat 3840 t at agcat t c gcagcact ct t cgcat ggag tt gat gggct gt gat t t aaa t agt t gcagc 3900

   Page 499

   2018203206 08 May 2018

   at gccat t gg gaat ggagag t aaagcaat a 97047_1 t cagat gcac agat t act gc t t cat cct ac 3960 t t t accaat a t gt t t gccac ct ggt ct cct t caaaagct c gact t cacct ccaagggagg 4020 agt aat gcct ggagacct ca ggt gaat aat ccaaaagagt ggct gcaagt ggact t ccag 4080 aagacaat ga aagt cacagg agt aact act cagggagt aa aat ct ct gct t accagcat g 4140 t at gt gaagg agt t cct cat ct ccagcagt caagat ggcc at cagt ggac t ct ct t t t t t 4200 cagaat ggca aagt aaaggt 1111 caggga aat caagact cct t cacacc t gt ggt gaac 4260 t ct ct agacc caccgt t act gact cgct ac ct t cgaat t c acccccagag t t gggt gcac 4320 cagat t gccc t gaggat gga ggt t ct gggc t gcgaggcac aggacct ct a cgacaaaact 4380 cacacat gcc caccgt gccc agct ccagaa ct cct gggcg gaccgt cagt ct t cct ct t c 4440 cccccaaaac ccaaggacac cct cat gat c t cccggaccc ct gaggt cac at gcgt ggt g 4500 gt ggacgt ga gccacgaaga ccct gaggt c aagt t caact ggt acgt gga cggcgt ggag 4560 gt gcat aat g ccaagacaaa gccgcgggag gagcagt aca acagcacgt a ccgt gt ggt c 4620 agcgt cct ca ccgt cct gca ccaggact gg ct gaat ggca aggagt acaa gt gcaaggt c 4680 t ccaacaaag ccct cccagc ccccat cgag aaaaccat ct ccaaagccaa agggcagccc 4740 cgagaaccac aggt gt acac cct gccccca t cccgggat g agct gaccaa gaaccaggt c 4800 agcct gacct gcct ggt caa aggct t ct at cccagcgaca t cgccgt gga gt gggagagc 4860 aat gggcagc cggagaacaa ct acaagacc acgcct cccg t gt t ggact c cgacggct cc 4920

   Page 500

   2018203206 08 May 2018

   97047_1

   11 ct t cct ct acagcaagct caccgt ggac aagagcaggt ggcagcaggg gaacgtcttc t cat gct ccg t gat gcat ga ggctctgcac aaccact aca cgcagaagag cct ct ccct g t ct ccgggt a aat ga <210> 126 <211> 1684 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> pSYNFVI I I 010 <400> 126

   4980

   5040

   5055

   Met 1 Gl n I l e Gl u Leu 5 Ser Thr Cy s Phe Phe Leu 10 Cy s Leu Leu Ar g 15 Phe Cy s Phe Ser Al a Thr Ar g Ar g Ty r Ty r Leu Gl y Al a Val Gl u Leu Ser 20 25 30 Tr p As p Ty r Met Gl n Ser As p Leu Gl y Gl u Leu Pr o Val As p Al a Ar g 35 40 45 Phe Pr o Pr o Ar g Val Pr o Lys Ser Phe Pr o Phe As n Thr Ser Val Val 50 55 60 Ty r Lys Lys Thr Leu Phe Val Gl u Phe Thr As p Hi s Leu Phe As n I l e

   Page 501

   97047_1

   65 70 75 80

   2018203206 08 May 2018

   Al a Lys Pr o Ar g Pr o 85 Pr o Tr p Met Gl y Leu 90 Leu Gl y Pr o Thr I l e 95 Gl n Al a Gl u Val Tyr As p Thr Val Val Ile Thr Leu Lys As n Met Al a Ser 100 105 110 Hi s Pr o Val Ser Leu Hi s Al a Val Gl y Val Ser Tyr Tr p Lys Al a Ser 115 120 125 Gl u Gl y Al a Gl u Tyr As p As p Gl n Thr Ser Gl n Ar g Gl u Lys Gl u As p 130 135 140 As p Lys Val Phe Pr o Gl y Gl y Ser Hi s Thr Tyr Val Tr p Gl n Val Leu 145 150 155 160 Lys Gl u As n Gl y Pr o Met Al a Ser As p Pr o Leu Cys Leu Thr Tyr Ser 165 170 175 Tyr Leu Ser Hi s Val As p Leu Val Lys As p Leu As n Ser Gl y Leu I l e 180 185 190 Gl y Al a Leu Leu Val Cys Ar g Gl u Gl y Ser Leu Al a Lys Gl u Lys Thr 195 200 205

   Page 502

   97047_1

   2018203206 08 May 2018

   Gl n Thr Leu Hi s Lys Phe Ile 215 Leu Leu Phe Al a Val 220 Phe As p Gl u Gl y 210 Lys Ser Tr p Hi s Ser Gl u Thr Lys As n Ser Leu Met Gl n As p Ar g As p 225 230 235 240 Al a Al a Ser Al a Ar g Al a Tr p Pr o Lys Met Hi s Thr Val As n Gl y Tyr 245 250 255 Val As n Ar g Ser Leu Pr o Gl y Leu Ile Gl y Cys Hi s Ar g Lys Ser Val 260 265 270 Tyr Tr p Hi s Val Ile Gl y Met Gl y Thr Thr Pr o Gl u Val Hi s Ser I l e 275 280 285 Phe Leu Gl u Gl y Hi s Thr Phe Leu Val Ar g As n Hi s Ar g Gl n Al a Ser 290 295 300 Leu Gl u I l e Ser Pr o Ile Thr Phe Leu Thr Al a Gl n Thr Leu Leu Met 305 310 315 320 As p Leu Gl y Gl n Phe Leu Leu Phe Cys Hi s Ile Ser Ser Hi s Gl n Hi s 325 330 335 As p Gl y Met Gl u Al a Tyr Val Lys Val As p Ser Cys Pr o Gl u Gl u Pr o

   Page 503

   2018203206 08 May 2018

   970 47_1 340 345 350 Gl n Leu Ar g Met Lys As n As n Gl u Gl u Al a Gl u As p Ty r As p As p As p 355 360 365 Leu Thr As p Ser Gl u Met As p Val Val Ar g Phe As p As p As p As n Ser 370 375 380 Pr o Ser Phe I l e Gl n Ile Ar g Ser Val Al a Lys Lys Hi s Pr o Lys Thr 385 390 395 400 Tr p Val Hi s Ty r Ile Al a Al a Gl u Gl u Gl u As p Tr p As p Ty r Al a Pr o 405 410 415 Leu Val Leu Al a Pr o As p As p Ar g Ser Ty r Lys Ser Gl n Ty r Leu As n 420 425 430 As n Gl y Pr o Gl n Ar g Ile Gl y Ar g Lys Ty r Lys Lys Val Ar g Phe Met 435 440 445 Al a Ty r Thr As p Gl u Thr Phe Lys Thr Ar g Gl u Al a I l e Gl n Hi s Gl u 450 455 460 Ser Gl y I l e Leu Gl y Pr o Leu Leu Ty r Gl y Gl u Val Gl y As p Thr Leu 465 470 475 480

   Page 504

   97047_1

   2018203206 08 May 2018

   Leu I l e I l e Phe Lys 485 As n Gl n Al a Ser Ar g 490 Pro Tyr As n I l e Tyr 495 Pr o Hi s Gl y I l e Thr As p Val Ar g Pr o Leu Tyr Ser Ar g Ar g Leu Pr o Lys 500 505 510 Gl y Val Lys Hi s Leu Lys As p Phe Pr o Ile Leu Pr o Gl y Gl u I l e Phe 515 520 525 Lys Tyr Lys Tr p Thr Val Thr Val Gl u As p Gl y Pr o Thr Lys Ser As p 530 535 540 Pr o Ar g Cys Leu Thr Ar g Tyr Tyr Ser Ser Phe Val As n Met Gl u Ar g 545 550 555 560 As p Leu Al a Ser Gl y Leu Ile Gl y Pr o Leu Leu Ile Cys Tyr Lys Gl u 565 570 575 Ser Val As p Gl n Ar g Gl y As n Gl n Ile Met Ser As p Lys Ar g As n Val 580 585 590 I l e Leu Phe Ser Val Phe As p Gl u As n Ar g Ser Tr p Tyr Leu Thr Gl u 595 600 605 As n I l e Gl n Ar g Phe Leu Pr o As n Pr o Al a Gl y Val Gl n Leu Gl u As p

   Page 505

   2018203206 08 May 2018

   97047_1

   610 615 620

   Pr o 625 Gl u Phe Gl n Al a Ser 630 As n Ile Met Hi s Ser 635 Ile As n Gl y Ty r Val 640 Phe As p Ser Leu Gl n Leu Ser Val Cy s Leu Hi s Gl u Val Al a Ty r Tr p 645 650 655 Ty r I l e Leu Ser Ile Gl y Al a Gl n Thr As p Phe Leu Ser Val Phe Phe 660 665 670 Ser Gl y Ty r Thr Phe Lys Hi s Lys Met Val Ty r Gl u As p Thr Leu Thr 675 680 685 Leu Phe Pr o Phe Ser Gl y Gl u Thr Val Phe Met Ser Met Gl u As n Pr o 690 695 700 Gl y Leu Tr p I l e Leu Gl y Cy s Hi s As n Ser As p Phe Ar g As n Ar g Gl y 705 710 715 720 Met Thr Al a Leu Leu Lys Val Ser Ser Cy s As p Lys As n Thr Gl y As p 725 730 735 Ty r Ty r Gl u As p Ser Ty r Gl u As p Ile Ser Al a Ty r Leu Leu Ser Lys 740 745 750

   Page 506

   97047_1

   2018203206 08 May 2018

   As n As n Al a 755 I l e Gl u Pr o Ar g Ser 760 Phe Ser Gl n As n Pr o 765 Pr o Val Leu Lys Ar g Hi s Gl n Ar g Gl u Ile Thr Ar g Thr Thr Leu Gl n Ser As p Gl n 770 775 780 Gl u Gl u I l e As p Tyr As p As p Thr Ile Ser Val Gl u Met Lys Lys Gl u 785 790 795 800 As p Phe As p I l e Tyr As p Gl u As p Gl u As n Gl n Ser Pr o Ar g Ser Phe 805 810 815 Gl n Lys Lys Thr Ar g Hi s Tyr Phe Ile Al a Al a Val Gl u Ar g Leu Tr p 820 825 830 As p Tyr Gl y Met Ser Ser Ser Pr o Hi s Val Leu Ar g As n Ar g Al a Gl n 835 840 845 Ser Gl y Ser Val Pr o Gl n Phe Lys Lys Val Val Phe Gl n Gl u Phe Thr 850 855 860 As p Gl y Ser Phe Thr Gl n Pr o Leu Tyr Ar g Gl y Gl u Leu As n Gl u Hi s 865 870 875 880 Leu Gl y Leu Leu Gl y Pr o Tyr Ile Ar g Al a Gl u Val Gl u As p As n I l e

   Page 507

   97047_1

   2018203206 08 May 2018

   885 890 895 Met Val Thr Phe Ar g As n Gl n Al a Ser Ar g Pr o Tyr Ser Phe Tyr Ser 900 905 910 Ser Leu I l e Ser Ty r Gl u Gl u As p Gl n Ar g Gl n Gl y Al a Gl u Pr o Ar g 915 920 925 Lys Asn Phe Val Lys Pro Asn Gl u Thr Lys Thr Tyr Phe Trp Lys Val 930 935 940 Gl n Hi s Hi s Met Al a Pr o Thr Lys Asp Gl u Phe Asp Cys Lys Al a Tr p 945 950 955 960 Al a Tyr Phe Ser Asp Val Asp Leu Gl u Ly s As p Val Hi s Ser Gl y Leu 965 970 975 I l e Gl y Pr o Leu Leu Val Cy s Hi s Thr As n Thr Leu As n Pr o Al a Hi s 980 985 990 Gl y Ar g Gl n Val Thr Val Gl n Gl u Phe Al a Leu Phe Phe Thr I l e Phe 995 1000 1005

   Asp Gl u Thr Lys Ser Trp Tyr Phe Thr Gl u Asn Met Gl u Arg Asn

   1010 1015 1020

   Page 508

   97047_1

   2018203206 08 May 2018

   Cys Ar g 1025 Al a Pro Cys Asn Ile 1030 Gl n Met Gl u As p Pr o 1035 Thr Phe Lys Gl u As n Tyr Ar g Phe Hi s Al a Ile As n Gl y Tyr Ile Met As p Thr 1040 1045 1050 Leu Pr o Gl y Leu Val Met Al a Gl n As p Gl n Ar g Ile Ar g Tr p Tyr 1055 1060 1065 Leu Leu Ser Met Gl y Ser As n Gl u As n Ile Hi s Ser I l e Hi s Phe 1070 1075 1080 Ser Gl y Hi s Val Phe Thr Val Ar g Lys Lys Gl u Gl u Tyr Lys Met 1085 1090 1095 Al a Leu Tyr As n Leu Tyr Pr o Gl y Val Phe Gl u Thr Val Gl u Met 1100 1105 1110 Leu Pr o Ser Lys Al a Gl y Ile Tr p Ar g Val Gl u Cys Leu I l e Gl y 1115 1120 1125 Gl u Hi s Leu Hi s Al a Gl y Met Ser Thr Leu Phe Leu Val Tyr Ser 1130 1135 1140 As n Lys Cys Gl n Thr Pr o Leu Gl y Met Al a Ser Gl y Hi s I l e Ar g

   Page 509

   2018203206 08 May 2018

   97047_1

   1145 1150 1155

   As p Phe 1160 Gl n Ile Thr Al a Ser 1165 Gl y Gl n Tyr Gl y Gl n 1170 Tr p Al a Pr o Lys Leu Al a Ar g Leu Hi s Tyr Ser Gl y Ser Ile As n Al a Tr p Ser 1175 1180 1185 Thr Lys Gl u Pr o Phe Ser Tr p Ile Lys Val As p Leu Leu Al a Pr o 1190 1195 1200 Met I l e I l e Hi s Gl y Ile Lys Thr Gl n Gl y Al a Ar g Gl n Lys Phe 1205 1210 1215 Ser Ser Leu Tyr Ile Ser Gl n Phe Ile Ile Met Tyr Ser Leu As p 1220 1225 1230 Gl y Lys Lys Tr p Gl n Thr Tyr Ar g Gl y As n Ser Thr Gl y Thr Leu 1235 1240 1245 Met Val Phe Phe Gl y As n Val As p Ser Ser Gl y Ile Lys Hi s As n 1250 1255 1260

   I l e Phe

   As n Pr o Pr o I l e I l e

   Al a Arg Tyr Ile Arg

   Leu Hi s Pr o

   1265

   1270

   1275

   Page 510

   97047_1

   2018203206 08 May 2018

   Thr Hi s 1280 Tyr Ser Ile Ar g Ser 1285 Thr Leu Ar g Met Gl u 1290 Leu Met Gl y Cys As p Leu As n Ser Cys Ser Met Pr o Leu Gl y Met Gl u Ser Lys 1295 1300 1305 Al a I l e Ser As p Al a Gl n Ile Thr Al a Ser Ser Tyr Phe Thr As n 1310 1315 1320 Met Phe Al a Thr Tr p Ser Pr o Ser Lys Al a Ar g Leu Hi s Leu Gl n 1325 1330 1335 Gl y Ar g Ser As n Al a Tr p Ar g Pr o Gl n Val As n As n Pr o Lys Gl u 1340 1345 1350 Tr p Leu Gl n Val As p Phe Gl n Lys Thr Met Lys Val Thr Gl y Val 1355 1360 1365 Thr Thr Gl n Gl y Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys 1370 1375 1380 Gl u Phe Leu Ile Ser Ser Ser Gl n As p Gl y Hi s Gl n Tr p Thr Leu 1385 1390 1395 Phe Phe Gl n As n Gl y Lys Val Lys Val Phe Gl n Gl y As n Gl n As p

   Page 511

   2018203206 08 May 2018

   9 7047 _1 1400 1405 1410 Ser Phe Thr Pr o Val Val As n Ser Leu As p Pr o Pr o Leu Leu Thr 1415 1420 1425 Ar g Tyr Leu Ar g Ile Hi s Pr o Gl n Ser Tr p Val Hi s Gl n I l e Al a 1430 1435 1440 Leu Ar g Met Gl u Val Leu Gl y Cys Gl u Al a Gl n As p Leu Tyr As p 1445 1450 1455 Lys Thr Hi s Thr Cys Pr o Pr o Cys Pr o Al a Pr o Gl u Leu Leu Gl y 1460 1465 1470 Gl y Pr o Ser Val Phe Leu Phe Pr o Pr o Lys Pr o Lys As p Thr Leu 1475 1480 1485 Met I l e Ser Ar g Thr Pr o Gl u Val Thr Cys Val Val Val As p Val 1490 1495 1500 Ser Hi s Gl u As p Pr o Gl u Val Lys Phe As n Tr p Tyr Val As p Gl y 1505 1510 1515 Val Gl u Val Hi s As n Al a Lys Thr Lys Pr o Ar g Gl u Gl u Gl n Tyr 1520 1525 1530

   Page 512

   97047_1

   2018203206 08 May 2018

   As n Ser 1535 Thr Tyr Ar g Val Val 1540 Ser Val Leu Thr Val 1545 Leu Hi s Gl n As p Tr p Leu As n Gl y Lys Gl u Tyr Lys Cys Lys Val Ser As n Lys 1550 1555 1560 Al a Leu Pr o Al a Pr o Ile Gl u Lys Thr Ile Ser Lys Al a Lys Gl y 1565 1570 1575 Gl n Pr o Ar g Gl u Pr o Gl n Val Tyr Thr Leu Pr o Pr o Ser Ar g As p 1580 1585 1590 Gl u Leu Thr Lys As n Gl n Val Ser Leu Thr Cys Leu Val Lys Gl y 1595 1600 1605 Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n 1610 1615 1620 Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Leu As p Ser As p 1625 1630 1635 Gl y Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val As p Lys Ser Ar g 1640 1645 1650 Tr p Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Met Hi s Gl u Al a

   Page 513

   2018203206 08 May 2018

   97047_1

   1655 1660 1665

   Leu Hi s As n Hi s Ty r Thr Gl n Ly s Ser Leu Ser Leu Ser Pr o Gl y 1670 1675 1680

   Lys <210> 127 <211> 78 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE72 XTEN <400> 127

   Gl y Al a Pr o Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser

   1 5 10 15

   Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a

   20 25 30

   Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u

   Thr

   Pr o Gl y Thr Ser

   Gl u Ser Al a

   Thr

   Pr o Gl u Ser

   Gl y Pr o Gl y Thr

   Page 514

   2018203206 08 May 2018

   97047_1

   50 55 60

   Ser Thr Gl u Pr o Ser Gl u Gl y Ser 65 70 <210> 128 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE144_2A <400> 128

   Thr Ser Thr Gl u Pr o Ser Gl u Gl y 1 5

   Ser Pr o Thr Ser Thr Gl u Gl u Gl y 20

   Ser Al a Pr o Gl y Thr Ser Thr Gl u 35 40

   Thr Ser Gl u Ser Al a Thr Pr o Gl u

   50 55

   Pr o Ser Gl u Gl y Ser Al a Pr o Gl y

   Al a Pr o Gl y Al a Ser Ser 75

   Ser Al a Pr o Gl y Ser Pr o Al a Gl y 10 15

   Thr Ser Thr Gl u Pr o Ser Gl u Gl y 25 30

   Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 45

   Ser Gl y Pr o Gl y Thr Ser Thr Gl u 60

   Thr Ser Gl u Ser Al a Thr Pr o Gl u

   Page 515

   2018203206 08 May 2018

   970 47_1 65 70 75 Ser Gl y Pr o Gl ^y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o 85 90 95 Thr Ser Thr Gl u Pr o Ser Gl u Gl ^y Ser Al a Pr o Gl y Thr Ser Thr 10 0 105 110 Pr o Ser Gl u Gl ^y Ser Al a Pr o Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 115 12 0 125 Ser Gl y Pr o Gl ^y Thr Ser Gl u Se r Al a Thr Pr o Gl u Ser Gl y Pr o

   130 135 140

   Gl y

   Gl u

   Gl u

   Gl y <210> 129 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE144_3B <400> 129 Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u 1 5 10 15

   Ser

   Al a Thr

   Pr o Gl u Ser Gl y Pr o Gl y

   Ser

   Gl u Pr o Al a

   Thr Ser Gl y

   Ser

   Page 516

   2018203206 08 May 2018

   97047_1

   20 25 30

   Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 35 40 45

   Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr Gl u 50 55 60

   Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y 65 70 75 80

   Ser Al a Pr o Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y 85 90 95

   Thr Ser Thr

   Gl u Pr o Ser

   100

   Gl u Gl y Ser

   Al a Pr o Gl y Thr

   Ser Thr

   Gl u

   105

   110

   Pr o Ser

   Gl u Gl y Ser 115

   Al a Pr o Gl y Ser 120

   Pr o Al a Gl y Ser Pr o Thr Ser 125

   Thr Gl u Gl u Gl y Thr

   Ser

   Thr

   Gl u Pr o Ser

   130

   135

   Gl u Gl y Ser 140

   Al a Pr o Gl y

   <210> 130 <211> 144 <212> PRT

   Page 517

   97047_1

   2018203206 08 May 2018 <213> Ar t i f i ci al Sequence <220>

   <223> AE144_4A <400> 130

   Thr Ser Gl u Ser Al a Thr Pr o 1 5 Thr Ser Gl y Ser Gl u Thr Pr o 20 Ser Gl y Pr o Gl y Ser Gl u Pr o 35 Thr Ser Gl u Ser Al a Thr Pr o 50 55 Pr o Ser Gl u Gl y Ser Al a Pr o 65 70 Ser Gl y Pr o Gl y Ser Pr o Al a 85 Ser Pr o Al a Gl y Ser Pr o Thr

   100

   Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 10 15 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 25 30 Al a Thr Ser Gl y Ser Gl u Thr Pr o 40 45 Gl u Ser Gl y Pr o Gl y Thr Ser Thr 60 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 75 Gl ^y Ser Pr o Thr Ser Thr Gl u Gl u 90 95 Se r Thr Gl u Gl u Gl y Ser Pr o Al a 105 110

   Al a

   Gl u

   Gl y

   Gl u

   Gl u

   Gl y

   Gl y

   Page 518

   2018203206 08 May 2018

   Ser Pr o Thr 115 Ser Thr Ser Gl y 130 Pr o Gl y Thr

   97047 1

   Gl u Gl u Gl y 120 Thr Ser Ser Thr Gl u Pr o Ser 135

   Gl u Ser Al a Thr

   125

   Gl u Gl y Ser Al a 140

   Pr o Gl u

   Pr o Gl y <210> 131 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE144_5A <400> 131

   Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u 1 5 10

   Pr o Al a

   Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr

   Pr o Gl u

   Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr

   Pr o Gl y

   Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Thr Ser

   Thr Gl u

   Page 519

   97047_1

   2018203206 08 May 2018

   Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 65 70 75 80 Thr Gl u Gl u Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y 85 90 95 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser 100 105 110 Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser 115 120 125 Thr Gl u Gl u Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y 130 135 140 <210> 132 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220> <223> AE144 _6B <400> 132 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Thr Ser Gl u Ser 1 5 10 15

   Page 520

   97047_1

   2018203206 08 May 2018

   Al a Thr Pr o Gl u 20 Ser Gl y Pr o Gl y Thr 25 Ser Gl u Ser Al a Thr 30 Pr o Ser Gl y Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o Gl u Ser Gl y Pr o 35 40 45 Ser Gl u Pr o Al a Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Gl u Pr o 50 55 60 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Ser Pr o Al a Gl y Ser Pr o Thr 65 70 75 Thr Gl u Gl u Gl y Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 85 90 95 Thr Ser Thr Gl u Pr o Ser Gl u Gl y Ser Al a Pr o Gl y Ser Gl u Pr o 100 105 110 Thr Ser Gl y Ser Gl u Thr Pr o Gl y Thr Ser Gl u Ser Al a Thr Pr o 115 120 125

   Gl u

   Gl y

   Al a

   Ser

   Gl y

   Al a

   Gl u

   Ser Gl y Pr o Gl y Thr Ser Thr

   Gl u Pr o Ser

   Gl u Gl y Ser Al a Pr o

   Gl y

   130

   135

   140 <210> 133

   Page 521

   2018203206 08 May 2018

   97047_1 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AG144_A <400> 133

   Gl y 1 Al a Ser Pr o Gl y 5 Thr Ser Ser Thr Gl y 10 Ser Pr o Gl y Ser Ser 15 Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr 20 25 30 Gl y Thr Gl y Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o 35 40 45 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o 50 55 60 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 65 70 75 80 Thr Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o 85 90 95 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Thr Pr o Gl y

   Page 522

   97047_1

   2018203206 08 May 2018

   100 105 110 Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 115 120 125 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 130 135 140

   <210> 134 <211> 144 <212> PRT <213> Artificial Sequence <220>

   <223> AG144_B <400> 134

   Gl y 1 Thr Pr o Gl y Ser 5 Gl y Thr Al a Ser Ser 10 Ser Pr o Gl y Ser Ser 15 Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 20 25 30 Thr Gl y Ser Pr o Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o 35 40 45 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o

   Page 523

   97047_1

   2018203206 08 May 2018

   50 55 60 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr 65 70 75 80 Gl y Thr Gl y Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o 85 90 95 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Al a Ser Pr o 100 105 110 Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 115 120 125 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 130 135 140

   <210> 135 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AG144_C <400> 135

   Gl y Thr Pr o Gl y Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o

   Page 524

   2018203206 08 May 2018

   97047_1

   1 5 10 15

   Gl y Thr Ser Ser 20 Thr Gl y Ser Pr o Gl y 25 Al a Ser Pr o Gl y Thr 30 Ser Ser Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 35 40 45 Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Thr Pr o Gl y 50 55 60 Ser Gl y Thr Al a Ser Ser Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 65 70 75 80 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 85 90 95 Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o Gl y Ser Ser Thr 100 105 110 Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Thr Pr o Ser Gl y Al a 115 120 125 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 130 135 140

   Page 525

   97047_1

   2018203206 08 May 2018 <210> 136 <211> 144 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AG144_F <400> 136

   Gl y 1 Ser Ser Pr o Ser 5 Al a Ser Thr Gl y Thr 10 Gl y Pr o Gl y Ser Ser 15 Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 20 25 30 Thr Gl y Ser Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser Thr Gl y Ser Pr o 35 40 45 Gl y Ser Ser Thr Pr o Ser Gl y Al a Thr Gl y Ser Pr o Gl y Ser Ser Pr o 50 55 60 Ser Al a Ser Thr Gl y Thr Gl y Pr o Gl y Al a Ser Pr o Gl y Thr Ser Ser 65 70 75 80 Thr Gl y Ser Pr o Gl y Ser Ser Pr o Ser Al a Ser Thr Gl y Thr Gl y Pr o 85 90 95

   Page 526

   97047_1

   2018203206 08 May 2018

   Gl y Thr

   Pr o Gl y Ser

   100

   Pr o Ser Gl y Al a Thr

   115

   Thr Gl y Ser

   130

   Gl y Thr Al a Ser

   Gl y Ser

   Pr o Gl y Al a Ser

   135

   Ser

   Ser

   Pr o Gl y Ser Ser Thr

   105

   110

   Pr o Gl y Ser Ser Thr

   120

   Pr o Gl y Thr Ser

   Pr o Ser Gl y Al a

   125

   Ser Thr

   Gl y Ser

   Pr o

   140 <210> 137 <211> 288 <212> PRT <213> Art i f i ci al Sequence <220>

   <223> AE288_2 <400> 137

   Gl y Ser Pr o Al a Gl y Ser Pr o Thr Ser Thr Gl u Gl u Gl y Thr Ser Gl u 1 5 10 15

   Ser Al a Thr Pr o Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o Al a Thr Ser Gl y

   Ser Gl u Thr

   Pr o Gl y Thr

   Ser Gl u Ser

   Al a Thr Pr o

   Gl u Ser Gl y Pr o 45

   Page 527

   2018203206 08 May 2018

   Gl y Thr 50 Ser Thr Gl u Pr o Ser 55 Gl u 65 Pr o Ser Gl u Gl y Ser 70 Al a Gl y Ser Al a Pr o Gl y 85 Thr Ser Gl y Thr Ser Thr 100 Gl u Pr o Ser Gl u Pr o Ser 115 Gl u Gl y Ser Al a Ser Thr 130 Gl u Gl u Gl y Thr Ser 135 Gl y 145 Thr Ser Gl u Ser Al a 150 Thr Al a Thr Ser Gl y Ser 165 Gl u Thr Gl u Ser Gl y Pr o Gl y Ser Gl u

   97047_1

   Gl y Ser Al a Pr o Gl y Thr Ser Thr 60

   Gl y Thr Ser Thr Gl u Pr o Ser Gl u 75 80

   Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 90 95

   Gl y Ser Al a Pr o Gl y Thr Ser Thr 105 110

   Gl y Ser Pr o Al a Gl y Ser Pr o Thr 125

   Gl u Pr o Ser Gl u Gl y Ser Al a Pr o 140

   Gl u Ser Gl y Pr o Gl y Ser Gl u Pr o 155 160

   Gl y Thr Ser Gl u Ser Al a Thr Pr o 170 175

   Al a Thr Ser Gl y Ser Gl u Thr Pr o

   Page 528

   97047_1

   2018203206 08 May 2018

   180 Gl y Thr Ser Gl u Ser Al a Thr Pr o 195 200 Gl u Pro Ser Gl u Gl y Ser Al a Pr o 210 215 Gl u Ser Gl y Pr o Gl y Ser Pr o Al a 225 230 Gl y Ser Pr o Al a Gl y Ser Pr o Thr 245 Gl y Ser Pr o Thr Ser Thr Gl u Gl u 260 Gl u Ser Gl y Pr o Gl y Thr Ser Thr 275 280 <210> 138 <211> 20 <212> PRT <213> Art i f i ci al Sequence <220> <223> l i nker

   18 5 190 Gl u Ser Gl y Pr o Gl y Thr Ser Thr 205 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 22 0 Gl ^y Ser Pr o Th r Ser Thr Gl u Gl u 235 240 Se r Thr Gl u Gl u Gl y Ser Pr o Al a 250 255 Gl ^y Thr Ser Gl u Ser Al a Thr Pr o 26 5 270 Gl u Pr o Ser Gl u Gl y Ser Al a Pr o

   285

   Page 529

   97047 1

   2018203206 08 May 2018

   <400> 138 Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y Gl y Gl y Gl y Ser Gl y 1 5 10 15

   Gl y Gl y Gl y Ser 20

   <210> 139 <211> 5 <212> PRT <213> Art i f i ci al Sequence

   <220> <223> l i nker

   <220> <221> REPEAT <222> (1). . (5) <223> May be repeated 1 to 10 times <400> 139 Gl y Gl y Gl y Gl y Ser 1 5

   <210> 140 <211> 6 <212> PRT

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   97047_1 <213> Art i f i ci al Sequence <220>

   <223> l i nker <220>

   <221> REPEAT <222> (2)..(6) <223> May be r epeat ed 1 t o 10 t i mes <400> 140

   Ser Gl y Gl y Gl y Gl y Ser

   1 5

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